Reversible covalent linkage of functional molecules

ABSTRACT

The present invention relates to the use of a compound containing a moiety of formula (I) as a reagent for linking a compound of formula R 1 —H which comprises a first functional moiety of formula F 1  to a second functional moiety of formula F 2   
                         
wherein X, X′, Y, R 1 , F 1  and F 2  are as defined herein. The present invention also provides related processes and products. The present invention is useful for creating functional conjugate compounds, and specifically conjugates in which at least one of the constituent molecules carries a thiol group.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/389,625, filed Apr. 6, 2012, which is pending, and pursuant to 35U.S.C. § 371, application Ser. No. 13/389,625 is a National StageApplication of PCT/GB2010/001499, filed Aug. 9, 2010, which claimspriority to GB 0913967.6, filed Aug. 10, 2009, GB 0913965.0, filed Aug.10, 2009 and GB 0914321.5, filed Aug. 14, 2009. The entire contents ofeach of the aforementioned applications are incorporated herein byreference as if set forth in their entirety.

INTRODUCTION

It is well known that it can be desirable to link together two or moremolecules, which each have specific functional properties. In this way,it becomes possible to generate new molecules, known as conjugates,which have the combined characteristics of their components. Thistechnique provides an attractive means for modifying the existingproperties of functionally useful molecules, or adding entirely newfunctional aspects to such molecules, in a controlled and broadlyapplicable manner.

The possibility of conjugating together two or more functional compoundshas stimulated particularly strong interest in the biotechnologicalfield. Conjugation of biomolecules such as proteins or biologicallyactive molecules such as drugs to a secondary functional compound hasbeen used in a vast range of applications, including detectiontechniques, proteomics studies, purification methods and the diagnosisand treatment of disease. Such is the ubiquity of these methodologiesthat standard text books devoted entirely to this topic are nowavailable. One such textbook is “Bioconjugate Techniques” (Greg T.Hermanson, Academic Press Inc., 1996), the content of which is hereinincorporated by reference in its entirety.

Methods for joining together diverse functional compounds typicallyfocus on the use of relatively small cross-linker molecules. A linkingreagent of this type contains at least two functional groups. Each ofthese functional groups is capable of reacting with a functionalmolecule in order to generate a final, cross-linked conjugate molecule.

A very wide variety of functional groups for cross-linker reagents havebeen developed to react with specific target functional groups presenton the functional molecules that are to be joined together. For example,cross-linkers containing activated ester groups such as theN-hydroxysuccinimide esters have long been used to react with functionalmoieties containing reactive amine groups, such as proteins.Hydrazide-containing cross-linkers (for example, adipic aciddihydrazide) have been used for functionalising carboxyl-containingfunctional molecules such as glycoproteins.

Cross-linking of one functional molecule to another can also be achievedby targeting a reactive thiol group in a functional molecule. Thisapproach can be particularly attractive where the functional molecule inquestion is a peptide or a protein. One reason for this is thatthiol-containing cysteine residues typically have low natural abundancein proteins, thus opening up the possibility of highly selectivemodification procedures. Standard site-directed mutagenesis techniquesalso allow for the easy insertion of a cysteine residue at a specificlocation in a protein, so generating a reactive thiol group, which canthen be modified by a functional moiety via a suitable cross-linker.

Various compounds have been used as linking reagents capable of reactingwith a thiol group. These reagents include 1,2-dicarbonyl ethenederivatives, α-halo carbonyl compounds and thiosulfonates. Of these,1,2-dicarbonyl ethene derivatives, such as maleimides, are generallyrecognised to be the most selective reagents for reaction with a thiol,in particular with a cysteine moiety. The thiol group in athiol-containing functional molecule (“R—SH”) reacts with maleimide toproduce a thioether linkage as follows:

The reactive amide moiety in the maleimide is available to react with afurther functional compound, to yield a conjugate in which thethiol-containing functional molecule is attached to a further functionalmoiety. Maleimide reagents are therefore useful for conjugatingcysteine-containing proteins to various secondary molecules (forexample, a fluorophore, biotin, a polyethylene glycol or acarbohydrate). These secondary molecules are often joined to themaleimide ring by way of a chemically inert linker species.

Unfortunately, however, this approach to conjugate generation hasseveral disadvantages. For example, chemical manipulation of thereaction product is typically possible only at the amide moiety. Thismeans that it can be difficult to add more than one further functionalgroup to the thiol-containing compound with a single maleimide linker.Furthermore, the thioether bond formed between the thiol-containingfunctional molecule and the maleimide cross-linker is irreversible.Accordingly, the derivitisation effected via the cross-linker ispermanent and it is not possible to regenerate the nativethiol-containing reagent.

The irreversibility of the known derivisation reaction between athiol-containing reagent and a maleimide-containing cross-linker canplace severe limitations on its practical utility in areas such asprotein purification, quantitative proteomic analysis, probing bindingsites, enabling structural studies and in drug delivery. For example, ina purification method involving generation of a conjugate between athiol-containing protein and an affinity tag (such as biotin), it wouldnot be possible to regenerate the native protein after purification bydetachment from the maleimide. An inability to regeneratethiol-containing reagents can also be a serious problem when carryingout procedures involving proteins that are difficult to express, such asmany GPCRs (G-protein coupled receptors). The irreversibility of thecross-linking process also precludes the exploitation of bioconjugatemethodology in areas where lability of the bond between the cross-linkerand the protein is important (for example, where the cross-linkingentity is designed to block the activity of an enzyme for only alimited, and preferably controllable, period).

The present invention is based on the surprising finding that it isadvantageous to incorporate an electrophilic leaving group onto the C═Cdouble bond of a known 1,2-dicarbonyl ethene cross-linking reagent. Thatchemical modification enables a thiol-containing functional moiety suchas a peptide or a protein to link to the cross-linking reagent whileretaining the C═C double bond. This has the following advantages:

-   -   The reaction between the cross-linker and the thiol compound can        often be carried out rapidly and with high yield using only a        substantially stoichiometric amount of cross-linker.    -   The thioether bond between the cross-linker and the        thiol-containing molecule is readily reversible, and in        particular can be cleaved in a controlled manner at a time        chosen by the skilled worker.    -   The retention of the double bond in the compound obtained after        linking the thiol-containing functional moiety to the        cross-linker constitutes a reaction site for linking to further        functional compounds. It can therefore be easier to add extra        functional moieties to the conjugate.

The new cross-linking methodology is readily applicable across the fullspectrum of known methods involving conjugation of functional moieties,which are now routinely carried out in the art.

U.S. Pat. No. 4,680,272 describes the use of halogenated maleimides andderivatives thereof as a fluorescent “stain” for detecting proteinshaving amine or thiol groups. U.S. Pat. No. 4,680,272 does not, however,disclose the use of 1,2-dicarbonyl ethene cross-linking reagents havingan electrophilic leaving group on the C═C double bond for constructingconjugate molecules, nor that the bond formed between a thiol compoundand such a cross-linker is readily reversible and can often be carriedout at high yield using a substantially stoichiometric amount ofcross-linker.

Hong et al. (J. Am. Chem. Soc., 2009, 131 (29), pp 9986-9994) describesa new class of fluorogenic probes for thiols based on a7-oxanorbornadiene framework. In one specific experiment described inthis paper, a 7-oxanorbornadiene reagent carrying a dansyl fluorogenicmoiety was reacted with bovine serum albumin. The resulting productunderwent a retro-Diels-Alder reaction to generate a product comprisinga maleimide cross-linking moiety which carried the bovine serum albuminat one carbon atom of the C═C double bond and a hydrogen atom at theother carbon atom of the C═C double bond. Hong et al. does not, however,describe the use of 1,2-dicarbonyl ethenes carrying an electrophilicleaving group as reagents for constructing a conjugate molecule.

U.S. Pat. No. 7,504,430 B2 and Kar et al. (Mol. Cancer Ther 2006; 5(6)June 2006 pp 1511-1519) describe a process for makingmaleimide-containing pharmaceutical compounds where a3,4-dibromomaleimide derivative is reacted with small, optionallysubstituted mercaptoalkyl compounds. These documents do not, however,describe processes for constructing conjugate molecules comprising atleast two functional moieties as defined herein, nor that the bondformed between a thiol compound and a cross-linker according to thepresent invention is readily reversible and can often be carried out athigh yield using a substantially stoichiometric amount of cross-linker.

SUMMARY OF THE INVENTION

The present invention provides (1) use of a compound containing a moietyof formula (I) as a reagent for linking a compound of formula R₁—H whichcomprises a first functional moiety of formula F₁ to a second functionalmoiety of formula F₂

wherein:

-   -   X and X′ are the same or different and each represents oxygen,        sulfur or a group of formula ═NQ, in which Q is hydrogen,        hydroxyl, C₁₋₆ alkyl or phenyl;    -   Y is an electrophilic leaving group;    -   R₁ is a group of formula —F₁ or -L-F₁, wherein L is a linker        group, and R₁—H comprises at least a first SH group; and    -   the first functional moiety and the second functional moiety are        the same or different and are each selected from a detectable        moiety, an enzymatically active moiety, an affinity tag, a        hapten, an immunogenic carrier, an antibody or antibody        fragment, an antigen, a ligand, a biologically active moiety, a        liposome, a polymeric moiety, an amino acid, a peptide, a        protein, a cell, a carbohydrate, a DNA and an RNA;        wherein the group R₁ becomes attached to the moiety of        formula (I) via nucleophilic attack of the first SH group in the        compound of formula R₁—H at the 2-position of the moiety of        formula (I), such that the group Y at the 2-position is replaced        by the group R₁.

The present invention also provides (2) a process for producing aconjugate, which process comprises

-   (i) reacting a compound containing a moiety of formula (I) with a    compound of formula R₁—H, thus producing a compound containing a    moiety of formula (II)

-   (ii) subsequently linking a moiety of formula F₂ to said moiety of    formula (II);    -   wherein step (i) involves attaching the group R₁ via        nucleophilic attack of the first SH group in the compound of        formula R₁—H at the 2-position of the moiety of formula (I),        such that the group Y at the 2-position is replaced by the group        R₁,    -   and wherein X, X′, Y, R₁, F₂ and the compound containing a        moiety of formula (I) are all as defined in (1) above.

The present invention further provides (3) a process for producing aconjugate, which process comprises reacting a compound of formula R₁—Hwith a compound comprising (a) a moiety of formula (I) and (b) at leastone moiety of formula F₂ linked thereto, wherein:

-   -   the moiety of formula (I) and F₂ are each as defined in (1)        above;    -   R₁, is as defined in (1) above; and    -   the process involves attaching the group R₁ via nucleophilic        attack of the first SH group in the compound of formula R₁—H at        the 2-position of the moiety of formula (I), such that the group        Y at the 2-position is replaced by the group R₁.

Still further, present invention provides (4) a process which comprises

(i) providing a compound comprising a moiety of formula (II); and

(ii) cleaving the bond between the group R₁ and the carbon atom at the2-position of the moiety of formula (II);

wherein:

-   -   R₁ is as defined in (1) above; and    -   the moiety of formula (II) is as defined in (2) above.

As will be evident to a skilled chemist, all of these uses and processesare linked by the finding that it is advantageous to incorporate anelectrophilic leaving group onto the C═C double bond of a knowndicarbonyl ethene cross-linking reagent. Further, many of theintermediates and products involved in these uses and processes arebelieved to novel. The present invention therefore also provides thefollowing embodiments (5) to (9).

The present invention provides (5) a compound of formula (IIa)

wherein:

-   -   either:        -   R_(3a) represents a group of formula R₃ or a group of            formula F₂ or -L(F₂)_(m)(Z)_(n-m) and R_(3a)′ independently            represents a group of formula R₃′ or a group of formula F₂            or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula —O— or            —N(R_(33a′)), wherein R_(33a′) represents a group of formula            R_(33′) or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula            —N(R_(33a′))—N(R_(33a′))—, wherein each R_(33a′) is the same            or different and represents a group of formula R_(33′) or a            group of formula F₂ or -L(F₂)_(m)(Z)_(n-m);    -   R_(2a) represents a group of formula R₂ or a group of formula F₂        or -L(F₂)_(m)(Z)_(n-m);    -   m is an integer having a value of from zero to n;    -   the compound of formula (IIa) comprises at least one group of        formula F₂;    -   F₂ is as defined in (1) above;    -   X and X′ are the same or different and each represents oxygen,        sulfur or a group of formula ═NQ, in which Q is hydrogen,        hydroxyl, C₁₋₆ alkyl or phenyl;    -   either:        -   R₃ and R₃′ are the same or different and each represents a            hydrogen atom or a group of formula E, Nu, -L(Z)_(n) or IG;            or        -   R₃ and R₃′ together form a group of formula —N(R_(33′)),            wherein R_(33′) represents a hydrogen atom or a group of            formula Y, Nu, -L(Z)_(n) or IG;    -   R₂ represents a hydrogen atom or a group of formula Y, Nu,        -L(Z)_(n) or IG;    -   each group of formula E and Y is the same or different and        represents an electrophilic leaving group;    -   each group of formula Nu is the same or different and represents        a nucleophile selected from —OH, —SH, —NH₂ and —NH(C₁₋₆ alkyl);    -   each group of formula L is the same or different and represents        a linker group;    -   each group of formula Z is the same or different and represents        a reactive group attached to a group of formula L which is        capable of reacting with a compound containing a second        functional moiety as defined in claim 1 such that said second        functional moiety becomes linked to said group of formula L;    -   n is 1, 2 or 3; and    -   each group of formula IG is the same or different and represents        a moiety which is a C₁₋₂₀ alkyl group, a C₂₋₂₀ alkenyl group or        a C₂₋₂₀ alkynyl group, which is unsubstituted or substituted by        one or more substituents selected from halogen atoms and        sulfonic acid groups, and in which (a) 0, 1 or 2 carbon atoms        are replaced by groups selected from C₆₋₁₀ arylene, 5- to        10-membered heteroarylene. C₃₋₇ carbocyclylene and 5- to        10-membered heterocyclylene groups, and (b) 0, 1 or 2 —CH₂—        groups are replaced by groups selected from —O—, —S—, —S—S—,        —C(O)— and —N(C₁₋₆ alkyl)- groups, wherein:    -   (i) said arylene, heteroarylene, carbocyclylene and        heterocyclylene groups are unsubstituted or substituted by one        or more substituents selected from halogen atoms and C₁₋₆ alkyl.        C₁₋₆ alkoxy, C₁₋₆ alkylthiol, —N(C₁₋₆ alkyl)(C₁₋₆ alkyl), nitro        and sulfonic acid groups; and    -   (ii) 0, 1 or 2 carbon atoms in said carbocyclylene and        heterocyclylene groups are replaced by —C(O)— groups.

Also provided by the present invention is (6) a compound of formula(IIb)

wherein:

-   -   R₁ is as defined in (1) above;    -   X and X′ are the same or different and each represents oxygen,        sulfur or a group of formula ═NQ, in which Q is hydrogen,        hydroxyl, C₁₋₆ alkyl or phenyl;    -   either:        -   R₃ and R₃′ are the same or different and each represents a            hydrogen atom or a group of formula E, Nu, -L(Z)_(n) or IG;            or        -   R₃ and R₃′ together form a group of formula —O— or            —N(R_(33′)), wherein R_(33′) represents a hydrogen atom or a            group of formula Y, Nu, -L(Z)_(n) or IG; or        -   R₃ and R₃′ together form a group of formula            —N(R_(33′))—N(R_(33′))—, wherein each R_(33′) is the same or            different and represents a hydrogen atom or a group of            formula Y, Nu, -L(Z)_(n) or IG;    -   R₂ represents a hydrogen atom or a group of formula Y, Nu,        -L(Z)_(n) or IG;    -   each group of formula E and Y is the same or different and        represents an electrophilic leaving group;    -   each group of formula Nu is the same or different and represents        a nucleophile selected from —OH, —SH, —NH₂ and        -   —NH(C₁₋₆ alkyl);    -   each group of formula L is the same or different and represents        a linker group;    -   each group of formula Z is the same or different and represents        a reactive group attached to a group of formula I, which is        capable of reacting with a compound containing a second        functional moiety as defined in claim 1 such that said second        functional moiety becomes linked to said group of formula L;    -   n is 1, 2 or 3; and    -   each group of formula IG is the same or different and represents        a moiety which is a C₁₋₂₀ alkyl group, a C₂₋₂₀ alkenyl group or        a C₂₋₂₀ alkynyl group, which is unsubstituted or substituted by        one or more substituents selected from halogen atoms and        sulfonic acid groups, and in which (a) 0, 1 or 2 carbon atoms        are replaced by groups selected from C₆₋₁₀ arylene, 5- to        10-membered heteroarylene, C₃₋₇ carbocyclylene and 5- to        10-membered heterocyclylene groups, and (b) 0, 1 or 2 —CH₂—        groups are replaced by groups selected from —O—, —S—, —S—S—,        —C(O)— and —N(C₁₋₆ alkyl)- groups, wherein:    -   (i) said arylene, heteroarylene, carxocyclylene and        heterocyclylene groups are unsubstituted or substituted by one        or more substituents selected from halogen atoms and C₁₋₆ alkyl,        C₁₋₆ alkoxy, C₁₋₆ alkylthiol. —N(C₁₋₆ alkyl)(C₁₋₆ alkyl), nitro        and sulfonic acid groups; and    -   (ii) 0, 1 or 2 carbon atoms in said carbocyclylene and        heterocyclylene groups are replaced by —C(O)— groups;        provided that R₃ and R₃′ do not together form a group of formula        —N(R_(33′)).

The present invention further provides (7) a compound of formula (II)

wherein:

-   -   either:        -   R_(3a) represents a group of formula R₃ or a group of            formula F₂ or -L(F₂)_(m)(Z)_(n-m) and R_(3a)′ independently            represents a group of formula R₃′ or a group of formula F₂            or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula —O— or            —N(R_(33a′)), wherein R_(33a′) represents a group of formula            R_(33′) or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula            —N(R_(33a′))—N(R_(33a′))—, wherein each R_(33a′) is the same            or different and represents a group of formula R_(33′) or a            group of formula F₂ or -L(F₂)_(m)(Z)_(n-m);    -   R_(2a) represents a group of formula R₂ or a group of formula F₂        or -L(F₂)_(m)(Z)_(n-m);    -   m is an integer having a value of from zero to n;    -   R₁ is as defined in (1) above;    -   F₂ is as defined in (1) above;    -   X and X′ are the same or different and each represents oxygen,        sulfur or a group of formula ═NQ, in which Q is hydrogen,        hydroxyl, C₁₋₆ alkyl or phenyl;    -   either:        -   R₃ and R₃′ are the same or different and each represents a            hydrogen atom or a group of formula E, Nu, -L(Z)_(n) or IG;            or        -   R₃ and R₃′ together form a group of formula —O— or            —N(R_(33′)), wherein R_(33′) represents a hydrogen atom or a            group of formula Y, Nu, -L(Z)_(n) or IG; or        -   R₃ and R₃′ together form a group of formula            —N(R_(33′))—N(R_(33′))—, wherein each R_(33′) is the same or            different and represents a hydrogen atom or a group of            formula Y, Nu, -L(Z)_(n) or IG;        -   R₂ represents a hydrogen atom or a group of formula Y, Nu,            -L(Z)_(n) or IG;        -   each group of formula E and Y is the same or different and            represents an electrophilic leaving group;        -   each group of formula Nu is the same or different and            represents a nucleophile selected from —OH, —SH, —NH₂ and            -   —NH(C₁₋₆ alkyl)        -   each group of formula Z is the same or different and            represents a linker group;        -   each group of formula Z is the same or different and            represents a reactive group attached to a group of formula L            which is capable of reacting with a compound containing a            second functional moiety as defined in claim 1 such that            said second functional moiety becomes linked to said group            of formula L;        -   n is 1, 2 or 3; and        -   each group of formula IG is the same or different and            represents a moiety which is a C₁₋₂₀ alkyl group, a C₂₋₂₀            alkenyl group or a C₂₋₂₀ alkynyl group, which is            unsubstituted or substituted by one or more substituents            selected from halogen atoms and sulfonic acid groups, and in            which (a) 0, 1 or 2 carbon atoms are replaced by groups            selected from C₆₋₁₀ arylene, 5- to 10-membered            heteroarylene, C₃₋₇ carbocyclylene and 5- to 10-membered            heterocyclylene groups, and (b) 0, 1 or 2 —CH₂— groups are            replaced by groups selected from —O—, —S—, —S—S—, —C(O)— and            —N(C₁₋₆ alkyl)- groups, wherein:        -   (i) said arylene, heteroarylene, carbocyclylene and            heterocyclylene groups are unsubstituted or substituted by            one or more substituents selected from halogen atoms and            C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkylthiol, —N(C₁₋₆            alkyl)(C₁₋₆ alkyl), nitro and sulfonic acid groups; and        -   (ii) 0, 1 or 2 carbon atoms in said carbocyclylene and            heterocyclylene groups are replaced by —C(O)— groups;            which comprises at least one group of formula F₂ and in            which R_(2a) is not a hydrogen atom.

Still further, the present invention provides (8) a compound of formula(IIIa)

wherein:

-   -   either:        -   R_(3a) represents a group of formula R₃ or a group of            formula F₂ or -L(F₂)_(m)(Z)_(n-m) and R_(3a)′ independently            represents a group of formula R₃′ or a group of formula F₂            or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula —O— or            —N(R_(33a′)), wherein R_(33a′) represents a group of formula            R_(33′) or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula            —N(R_(33a′))—N(R_(33a′))—, wherein each R_(33a′) is the same            or different and represents a group of formula R_(33′) or a            group of formula F₂ or -L(F₂)_(m)(Z)_(n-m);    -   m is an integer having a value of from zero to n;    -   R₁ is as defined in (1) above, and wherein R₁ comprises at least        a first thiol group and a second thiol group, said first thiol        group being attached to the 2-position in the compound of        formula and second thiol group being attached to the 3-position        in the compound of formula (IIIa);    -   F₂ is as defined in (1) above;    -   X and X′ are the same or different and each represents oxygen,        sulfur or a group of formula ═NQ, in which Q is hydrogen,        hydroxyl, C₁₋₆ alkyl or phenyl;    -   either:        -   R₃ and R₃′ are the same or different and each represents a            hydrogen atom or a group of formula E, Nu, -L(Z)_(n) or IG;            or        -   R₃ and R₃′ together form a group of formula —O— or            —N(R_(33′)), wherein R_(33′) represents a hydrogen atom or a            group of formula Y, Nu, -L(Z)_(n) or IG; or        -   R₃ and R₃′ together form a group of formula            —N(R_(33′))—N(R_(33′))—, wherein each R_(33′) is the same or            different and represents a hydrogen atom or a group of            formula Y, Nu, -L(Z)_(n) or IG;    -   R₂ represents a hydrogen atom or a group of formula Y, Nu,        -L(Z)_(n) or IG;    -   each group of formula E and Y is the same or different and        represents an electrophilic leaving group;    -   each group of formula Nu is the same or different and represents        a nucleophile selected from —OH, —SH, —NH₂ and        -   —NH(C₁₋₆ alkyl);    -   each group of formula L is the same or different and represents        a linker group;    -   each group of formula Z is the same or different and represents        a reactive group attached to a group of formula I, which is        capable of reacting with a compound containing a second        functional moiety as defined in claim 1 such that said second        functional moiety becomes linked to said group of formula L;    -   n is 1, 2 or 3; and    -   each group of formula IG is the same or different and represents        a moiety which is a C₁₋₂₀ alkyl group, a C₂₋₂₀ alkenyl group or        a C₂₋₂₀ alkynyl group, which is unsubstituted or substituted by        one or more substituents selected from halogen atoms and        sulfonic acid groups, and in which (a) 0, 1 or 2 carbon atoms        are replaced by groups selected from C₆₋₁₀ arylene, 5- to        10-membered heteroarylene, C₃₋₇ carbocyclylene and 5- to        10-membered heterocyclylene groups, and (b) 0, 1 or 2 —CH₂—        groups are replaced by groups selected from —O—, —S—, —S—S—,        —C(O)— and —N(C₁₋₆ alkyl)- groups, wherein:    -   (i) said arylene, heteroarylene, carxocyclylene and        heterocyclylene groups are unsubstituted or substituted by one        or more substituents selected from halogen atoms and C₁₋₆ alkyl,        C₁₋₆ alkoxy, C₁₋₆ alkylthiol, —N(C₁₋₆ alkyl)(C₁₋₆ alkyl), nitro        and sulfonic acid groups; and    -   (ii) 0, 1 or 2 carbon atoms in said carbocyclylene and        heterocyclylene groups are replaced by —C(O)— groups.

The present invention provides (9) a compound of formula (IVa) or (IVb)

wherein

-   -   R₁ is as defined in (1) above;    -   X and X′ are the same or different and each represents oxygen,        sulfur or a group of formula ═NQ, in which Q is hydrogen,        hydroxyl, C₁₋₆ alkyl or phenyl;    -   either:        -   R_(3a) represents a group of formula R₃ or a group of            formula F₂ or -L(F₂)_(m)(Z)_(n-m) and R_(3a)′ independently            represents a group of formula R₃′ or a group of formula F₂            or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula —O— or            —N(R_(33a′)), wherein R_(33a′) represents a group of formula            R_(33′) or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula            —N(R_(33a′))—N(R_(33a′))—, wherein each R_(33a′) is the same            or different and represents a group of formula R_(33′) or a            group of formula F₂ or -L(F₂)_(m)(Z)_(n-m);    -   R_(2a) represents a group of formula R₂ or a group of formula F₂        or -L(F₂)_(m)(Z)_(n-m);    -   m is an integer having a value of from zero to n;    -   either:        -   R₃ and R₃′ are the same or different and each represents a            hydrogen atom or a group of formula E, Nu, -L(Z)_(n) or IG;            or        -   R₃ and R₃′ together form a group of formula —O— or            —N(R_(33′)), wherein R_(33′) represents a hydrogen atom or a            group of formula Y, Nu, -L(Z)_(n) or IG; or        -   R₃ and R₃′ together form a group of formula            —N(R_(33′))—N(R_(33′))—, wherein each R_(33′) is the same or            different and represents a hydrogen atom or a group of            formula Y, Nu, -L(Z)_(n) or IG;    -   R₂ represents a hydrogen atom or a group of formula Y, Nu,        -L(Z)_(n) or IG;        -   each group of formula E and Y is the same or different and            represents an electrophilic leaving group;        -   each group of formula Nu is the same or different and            represents a nucleophile selected from —OH, —SH, —NH₂ and            -   —NH(C₁₋₆ alkyl);        -   each group of formula L is the same or different and            represents a linker group;        -   each group of formula Z is the same or different and            represents a reactive group attached to a group of formula L            which is capable of reacting with a compound containing a            second functional moiety as defined in claim 1 such that            said second functional moiety becomes linked to said group            of formula L;        -   n is 1, 2 or 3;        -   each group of formula IG is the same or different and            represents a moiety which is a C₁₋₂₀ alkyl group, a C₂₋₂₀            alkenyl group or a C₂₋₂₀ alkynyl group, which is            unsubstituted or substituted by one or more substituents            selected from halogen atoms and sulfonic acid groups, and in            which (a) 0, 1 or 2 carbon atoms are replaced by groups            selected from C₆₋₁₀ arylene, 5- to 10-membered            heteroarylene, C₃₋₇ carbocyclylene and 5- to 10-membered            heterocyclylene groups, and (b) 0, 1 or 2 —CH₂— groups are            replaced by groups selected from —O—, —S—, —S—S—, —C(O)— and            —N(C₁₋₆ alkyl)- groups, wherein:        -   (i) said arylene, heteroarylene, carbocyclylene and            heterocyclylene groups are unsubstituted or substituted by            one or more substituents selected from halogen atoms and            C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkylthiol, —N(C₁₋₆            alkyl)(C₁₋₆ alkyl), nitro and sulfonic acid groups; and        -   (ii) 0, 1 or 2 carbon atoms in said carbocyclylene and            heterocyclylene groups are replaced by —C(O)— groups;    -   R₄ is a halogen atom, a hydroxyl, C₁₋₆ alkoxy, thiol, C₁₋₆        alkylthio or C₁₋₆ alkylcarbonyloxy group, or a group of formula        F₂;    -   at least one of the groups R_(2a) and R₄ comprises a group of        formula F₂; and    -   F₂ is as defined in (1) above.

The present invention also provides (10) a process for producing acompound of formula (IVa) or (IVb) as defined in (9) above, whichcomprises

-   (i) providing a compound of formula (III); and

-   (ii) reacting the compound of formula (III) with a compound of    formula R₄—H, wherein R₄ is as defined in (9) above;    wherein:    -   either:        -   R_(3a) represents a group of formula R₃ or a group of            formula F₂ or -L(F₂)_(m)(Z)_(n-m) and R_(3a)′ independently            represents a group of formula R₃′ or a group of formula F₂            or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula —O— or            —N(R_(33a′)), wherein R_(33a′) represents a group of formula            R_(33′) or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula            —N(R_(33a′))—N(R_(33a′))—, wherein each R_(33a′) is the same            or different and represents a group of formula R_(33′) or a            group of formula F₂ or -L(F₂)_(m)(Z)_(n-m);    -   R_(2a) represents a group of formula R₂ or a group of formula F₂        or -L(F₂)_(m)(Z)_(n-m);    -   m is an integer having a value of from zero to n;    -   R₁ is as defined in (1) above;    -   F₂ is as defined in (1) above;    -   X and X′ are the same or different and each represents oxygen,        sulfur or a group of formula ═NQ, in which Q is hydrogen,        hydroxyl, C₁₋₆ alkyl or phenyl;    -   either:        -   R₃ and R₃′ are the same or different and each represents a            hydrogen atom or a group of formula E, Nu, -L(Z)_(n) or IG;            or        -   R₃ and R₃′ together form a group of formula —O— or            —N(R_(33′)), wherein R_(33′) represents a hydrogen atom or a            group of formula Y, Nu, -L(Z)_(n) or IG; or        -   R₃ and R₃′ together form a group of formula            —N(R_(33′))—N(R_(33′))—, wherein each R_(33′) is the same or            different and represents a hydrogen atom or a group of            formula Y, Nu, -L(Z)_(n) or IG;    -   R₂ represents a hydrogen atom or a group of formula Y, Nu,        -L(Z)_(n) or IG;    -   each group of formula E and Y is the same or different and        represents an electrophilic leaving group;    -   each group of formula Nu is the same or different and represents        a nucleophile selected from —OH, —SH, —NH₂ and —NH(C₁₋₆ alkyl);    -   each group of formula L is the same or different and represents        a linker group;    -   each group of formula Z is the same or different and represents        a reactive group attached to a group of formula I, which is        capable of reacting with a compound containing a second        functional moiety as defined in claim 1 such that said second        functional moiety becomes linked to said group of formula L;    -   n is 1, 2 or 3; and    -   each group of formula IG is the same or different and represents        a moiety which is a C₁₋₂₀ alkyl group, a C₂₋₂₀ alkenyl group or        a C₂₋₂₀ alkynyl group, which is unsubstituted or substituted by        one or more substituents selected from halogen atoms and        sulfonic acid groups, and in which (a) 0, 1 or 2 carbon atoms        are replaced by groups selected from C₆₋₁₀ arylene, 5- to        10-membered heteroarylene, C₃₋₇ carbocyclylene and 5- to        10-membered heterocyclylene groups, and (b) 0, 1 or 2 —CH₂—        groups are replaced by groups selected from —O—, —S—, —S—S—,        —C(O)— and —N(C₁₋₆ alkyl)- groups, wherein:    -   (i) said arylene, heteroarylene, carxocyclylene and        heterocyclylene groups are unsubstituted or substituted by one        or more substituents selected from halogen atoms and C₁₋₆ alkyl,        C₁₋₆ alkoxy, C₁₋₆ alkylthiol, —N(C₁₋₆ alkyl)(C₁₋₆ alkyl), nitro        and sulfonic acid groups; and    -   (ii) 0, 1 or 2 carbon atoms in said carbocyclylene and        heterocyclylene groups are replaced by —C(O)— groups.

The present invention further provides (11) a process for detecting acompound of formula R₁—H in a sample, which comprises incubating saidsample with a compound comprising (a) a moiety of formula (I) and (b) acompound of formula F₂ linked thereto, under conditions allowing fordetection of said compound of formula R₁—H in said sample, wherein:

-   -   the compound comprising (a) a moiety of formula (I) and (b) a        compound of formula F₂ linked thereto is as defined in (3)        above; and    -   the compound of formula F₂ is a detectable moiety, which is        capable of producing a signal which can be modified by the group        of formula R₁.

Still further, the present invention provides (12) a process fordetecting whether a substance is present in a sample, which processcomprises:

-   -   providing a compound as defined in any one of (7), (8) and (9)        above, provided that the compound as defined in (8) comprises at        least one group of formula F₂, wherein one of said first        functional moiety and said second functional moiety is a        functional moiety that is capable of generating a detectable        signal and the other of said first functional moiety and said        second functional moiety is a functional moiety that is capable        of interacting with said substance;    -   incubating said sample with said compound; and    -   monitoring for a signal under conditions allowing for generation        of a detectable signal from said functional moiety that is        capable of generating a detectable signal.

The invention also provides (13) a process for identifying whether asubstance interacts with a functional moiety of formula R₁, whichprocess comprises:

-   -   producing a conjugate comprising (a) said functional moiety of        formula R₁, and (b) a detectable moiety which is capable of        producing a signal which can be modified by said substance, by        carrying out a process according to either of (2) and (3) above;    -   incubating said conjugate with said substance;    -   obtaining a signal from said detectable moiety; and    -   comparing said signal with a control signal obtainable when said        conjugate has not been contacted with the substance, thus        determining whether the substance interacts with the conjugate.

Furthermore, the present invention provides (14) a compound as definedin any one of (7), (8) and (9) above for use in a method of treatment ofthe human or animal body by surgery or therapy or a diagnostic methodpractised on the human or animal body, provided that the compound asdefined in (8) comprises at least one group of formula F₂.

The invention also provides a compound containing a moiety of formula(VI) and a functional moiety linked thereto

wherein:

-   -   X and X′ are the same or different and each represents oxygen,        sulfur or a group of formula ═NQ, in which Q is hydrogen,        hydroxyl, C₁₋₆ alkyl or phenyl; and    -   said functional moiety is selected from a detectable moiety, an        enzymatically active moiety, an affinity tag, a hapten, an        immunogenic carrier, an antibody or antibody fragment, an        antigen, a ligand, a biologically active moiety, a liposome, a        polymeric moiety, an amino acid, a peptide, a protein, a cell, a        carbohydrate, a DNA and an RNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of the protocol described in Example 31 whereinprotein/biotin-PEG-bromomaleimide adduct and unmodified model proteinsolutions (In) were added to neutravidin-coated agarose beads,centrifuged, the flow-through (FT) collected, the beads washed with PBSand both wash fractions collected (W1 and W2), protein released from thebeads by incubation in PBS containing β-mercaptoethanol, the samplecentrifuged and the eluant (EI) containing cleaved protein collected.

FIG. 2 shows the generation of somatostatin-maleimide adducts fromhalomaleimides according to the protocol described in Example 39 asmeasured by LC-MS (y-axis=signal %; x-axis=time/min). Top left:Generation of somatostatin adduct from dichloromaleimide (circle),dibromomaleimide (square) and diiodomaleimide (triangle). Top right:Generation of somatostatin adduct from monobromomaleimide (circle),N-methylmonobromomaleimide (square) and N-methyldibromomaleimide(triangle). Bottom left: Generation of somatostatin adduct fromN-fluorescein-dibromomaleimide (circle), N-biotin-dibmmomaleimide(square), N-PEG 5000-dibromomaleimide (triangle),N-PEG-5000-dithiophenolmaleimide (diamond) and N-PEG300-dibromomaleimide (oval).

FIG. 3 shows the generation of somatostatin-maleimide adducts fromdithiomaleimides according to the protocol described in Example 39 asmeasured by LC-MS (y-axis=signal % x-axis=time/min). Top left:Generation of somatostatin adduct from di-2-mercaptoethanolmaleinmide at1 eq. (circle), 5 eq. (square) and 10 eq. (triangle). Top right:Generation of somatostatin adduct from dicysteinemaleimide at 1 eq.(circle), 5 eq. (square) and 10 eq. (triangle). Bottom left: Generationof somatostatin adduct from dithiophenolmaleimide at 1 eq. (circle), 5eq. (square) and 10 eq. (triangle). Bottom right: Generation ofsomatostatin adduct from di-2-mercaptopyridinemaleimide at 1 eq.(circle), 5 eq. (square) and 10 eq. (triangle).

FIG. 4 shows cleavage of maleimide bridged somatostatin with variousreducing agents according to the protocol described in Example 39 asmeasured by LC-MS (y-axis=signal %; x-axis=time in minutes (min), hours(h) and days (d)). Top left: Total modified somatostatin-maleimide withDTT (hollow circle) and total amount of side products (filled circle).Top middle: Total modified somatostatin-maleimide with 2-mercaptoethanol(hollow circle) and total amount of side products (filled circle). Topright: Total modified somatostatin-maleimide with GSH (hollow circle)and total amount of side products (filled circle). Bottom left: Totalmodified somatostatin-maleimide with TCEP (hollow circle) and totalamount of side products (filled circle). Bottom right: Total modifiedsomatostatin-maleimide with 1,2-ethanedithiol (hollow circle) and totalamount of side products (filled circle).

FIG. 5 shows cleavage of maleimide bridged somatostatin with variousamounts of DTT and 2-mercaptoethanol according to the protocol describedin Example 39 as measured by LC-MS (y-axis=signal %; x-axis=time/min).Left: Regeneration of somatostatin by DTT at 50 eq. (hollow circle), 20eq. (hollow triangle) and 10 eq. (hollow square). Right: Regeneration ofsomatostatin by 2-mercaptoethanol at 50 eq. (hollow circle), 20 eq.(hollow triangle) and 10 eq. (hollow square) and total amount of sideproducts at 50 eq. (filled circle), 20 eq. (filled triangle) and 10 eq.(filled square).

FIG. 6 shows catalysed cleavage of bridged somatostatin according to theprotocol described in Example 39 as measured by LC-MS (y-axis=signal %;x-axis=time/min). Shown on the Figure are regeneration of somatostatinby 2-mercaptoethanol (hollow circle), 2-mercaptoethanol with NaI (hollowsquare) and 2-mercaptoethanol with benzeneselenol (hollow triangle), aswell as total side products when using 2-mercaptoethanol (filledcircle), 2-mercaptoethanol with NaI (filled square) and2-mercaptoethanol with benzeneselenol (filled triangle).

FIG. 7 shows cleavage of N-functionalised maleimide bridged somatostatinby 2-mercaptoethanol according to the protocol described in Example 39as measured by LC-MS (y-axis=signal %; x-axis=time in minutes (min),hours (h) and days (d)). Top left: cleavage of N-methylmaleimidesomatostatin adduct to give somatostatin (hollow circle) and total sideproducts (filled circle). Top middle: cleavage of N-biotin maleimidesomatostatin adduct to give somatostatin (hollow circle) and total sideproducts (filled circle). Top right: cleavage of N-fluorescein maleimidesomatostatin adduct to give somatostatin (hollow circle) and total sideproducts (filled circle). Bottom left: cleavage of N-PEG 5000 maleimidesomatostatin adduct to give somatostatin (hollow circle) and total sideproducts (filled circle). Bottom middle: cleavage of N-PEG 300 maleimidesomatostatin adduct to give somatostatin (hollow circle) and total sideproducts (filled circle).

FIG. 8 shows cleavage of the diaddition product of monobromomaleimidewith somatostatin according to the protocol described in Example 39 asmeasured by LC-MS (y-axis=signal %; x-axis=time/hours). Top left:Somatostatin-maleimide (hollow circle), somatostatin-bis-maleimide(filled circle) and total side products (triangle) using2-mercaptoethanol. Top right: Somatostatin-maleimide (hollow circle),somatostatin-bis-maleimide (filled circle) and total side products(triangle) using DTT. Bottom left: Somatostatin-maleimide (hollowcircle), somatostatin-bis-maleimide (filled circle) and total sideproducts (triangle) using TCEP.

FIG. 9 shows comparable in situ bridging of somatostatin with variousamounts of dithiomaleimides according to the protocol described inExample 39 as measured by LC-MS (y-axis=signal %; x-axis=time/min). TheFigure shows generation of bridged somatostatin using TCEP initiator andthiophenol in a ratio of 3:5 (circle), selenol initiator with thiophenolin a ratio of 5:10 (square) and selenol initiator with 2-mercaptoethanolin a ratio of 10:20 (triangle).

FIG. 10 shows in situ PEGylation of somatostatin according to theprotocol described in Example 39 as measured by LC-MS (y-axis=signal %;x-axis=time/min). The Figure shows generation of PEGylated somatostatinusing 5 eq. N-PEG5000-dithiophenolmaleimide and 3 eq. TCEP (circle) andusing 10 eq. N-PEG5000-dithiophenolmaleimide and 5 eq. benzeneselenol(square).

FIG. 11 shows whole cell patch-clamp current recordings obtained in thepatch clamp assay described in Example 39. The Figure showsrepresentative traces recorded from the (GIRK 1/2A cell line expressingSSTR2. The cells were clamped at −60 mV and 20 μM of somatostatin or itsderivatives were applied for 20 s. Top left: somatostatin (in the axesshown the vertical line represents 1000 pA and the horizontal linerepresents 20 ms). Top right: dibromomaleimide-bridged somatostatin (inthe axes shown the vertical line represents 1000 pA and the horizontalline represents 20 ms). Bottom left: fluoresceindibromomaleimide-bridged somatostatin (in the axes shown the verticalline represents 1000 pA and the horizontal line represents 20 ms).Bottom right: PEGylated dibromomaleimide-bridged somatostatin (in theaxes shown the vertical line represents 1000 pA and the horizontal linerepresents 20 ms).

FIG. 12 shows the amplitudes of the currents activated by somatostatinand its analogues in the patch clamp assay described in Example 39. Thex-axis represents current amplitude in pA/pF. Top two bars are fromfluorescein dibromomaleimide-bridged somatostatin (black bar is afterpre-treatment of cell with Pertussis toxin for 24 hr; grey bar is withno pre-treatment), next two bars are from PEGylateddibromomaleimide-bridged somatostatin (black bar is after pre-treatmentof cell with Pertussis toxin for 24 hr. grey bar is with nopre-treatment), next three bars are from dibromomaleimide-bridgedsomatostatin (white bar is after preincubation with the GIRK inhibitorTertiapinQ, 100 nM for 5 minutes; black bar is after pre-treatment ofcell with Pertussis toxin for 24 hr, grey bar is with no pre-treatment)and bottom two bars are from somatostatin (black bar is afterpre-treatment of cell with Pertussis toxin for 24 hr, grey bar is withno pre-treatment).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “functional moiety” means a moiety which formspart of a conjugate and which is one of a detectable moiety, anenzymatically active moiety, an affinity tag, a hapten, an immunogeniccarrier, an antibody or antibody fragment, an antigen, a ligand, abiologically active moiety, a liposome, a polymeric moiety, an aminoacid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA.

As will be readily understood by those of skill in the art, a functionalmoiety comprised within a compound (for example, within a conjugatemolecule) is obtainable by attaching a corresponding “functionalcompound” thereto. When a functional compound attaches to a secondarycompound, it is necessary for a bond somewhere in that functionalcompound to be broken so that a new bond can form to the secondarycompound. Examples of such processes include the loss of a leaving groupfrom the functional compound when it becomes a functional moiety boundto the secondary molecule, the loss of a proton when the functionalcompound reacts via a hydrogen-atom containing nucleophilic group suchas an —OH or —SH group, or the conversion of a double bond in thefunctional compound to a single bond when the functional compound reactswith the secondary compound via an electrophilic or nucleophilicadditional reaction. Those skilled in the art would thus understand thata functional moiety that is, for example, a “protein” means a moietythat is formed by incorporation of a protein compound into a secondarymolecule, with concomitant loss of a internal bond compared to thecorresponding protein compound (for example, loss of a proton from an—OH, —SH or —NH₂ moiety when such a moiety forms the bond to thesecondary molecule).

A functional moiety is typically a moiety that has a discrete biologicalsignificance in its native form (i.e., when it is not part of abioconjugate). Preferably any functional moiety used in the presentinvention has a relative molecular weight of at least 200, morepreferably at least 500, most preferably at least 1000. Preferably afunctional moiety as described herein is a biomolecule.

As used herein, the term “detectable moiety” means a moiety which iscapable of generating detectable signals in a test sample. Clearly, thedetectable moiety can be understood to be a moiety which is derived froma corresponding “detectable compound” and which retains its ability togenerate a detectable signal when it is linked to another functionalmoiety via a cross-linker in a conjugate of the present invention.Detectable moieties are also commonly known in the art as “tags”,“probes” and “labels”. Examples of detectable moieties includechromogenic moieties, fluorescent moieties, radioactive moieties andelectrochemically active moieties. In the present invention, preferreddetectable moieties are chromogenic moieties and fluorescent moieties.Fluorescent moieties are most preferred.

A chromogenic moiety is a moiety which is coloured, which becomescoloured when it is incorporated into a conjugate, or which becomescoloured when it is incorporated into a conjugate and the conjugatesubsequently interacts with a secondary target species (for example,where the conjugate comprises a protein which then interacts withanother target molecule).

Typically, the term “chromogenic moiety” refers to a group of associatedatoms which can exist in at least two states of energy, a ground stateof relatively low energy and an excited state to which it may be raisedby the absorption of light energy from a specified region of theradiation spectrum. Often, the group of associated atoms containsdelocalised electrons. Chromogenic moieties suitable for use in thepresent invention include conjugated moieties containing Π systems andmetal complexes. Examples include porphyrins, polyenes, polyynes andpolyaryls. Preferred chromogenic moieties are

A fluorescent moiety is a moiety which comprises a fluorophore, which isa fluorescent chemical moiety. Examples of fluorescent compounds whichare commonly incorporated as fluorescent moieties into secondarymolecules such as the conjugates of the present invention include:

-   -   the Alexa Fluor® dye family available from Invitrogen;    -   cyanine and merocyanine;    -   the BODIPY (boron-dipyrromethene) dye family, available from        Invitrogen;    -   the ATTO dye family manufactured by ATTO-TEC GmbH;    -   fluorescein and its derivatives;    -   rhodamine and its derivatives;    -   naphthalene derivatives such as its dansyl and prodan        derivatives;    -   pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole        derivatives;    -   coumarin and its derivatives;    -   pyrene derivatives; and    -   Oregon green, eosin, Texas red, Cascade blue and Nile red,        available from Invitrogen.

Preferred fluorescent moieties for use in the present invention includefluorescein, rhodamine, coumarin, sulforhodamine 101 acid chloride(Texas Red) and dansyl. Fluorescein and dansyl are especially preferred.

A radioactive moiety is a moiety that comprises a radionuclide. Examplesof radionuclides include iodine-131, iodine-125, bismuth-212,yttrium-90, yttrium-88, technetium-99m, copper-67, rhenium-188,rhenium-186, gallium-66, gallium-67, indium-111, indium-14m, indium-114,boron-10, tritium (hydrogen-3), carbon-14, sulfur-35, fluorine-18 andcarbon-11. Fluorine-18 and carbon-11, for example, are frequently usedin positron emission tomography.

In one embodiment, the radioactive moiety may consist of theradionuclide alone. In another embodiment, the radionuclide may beincorporated into a larger radioactive moiety, for example by directcovalent bonding to a linker group (such as a linker containing a thiolgroup) or by forming a co-ordination complex with a chelating agent.Suitable chelating agents known in the art include DTPA(diethylenetriamine-pentaacetic anhydride), NOTA(1,4,7-triazacyclononane-N,N′,N″-triacetic acid), DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid), TETA(1,4,8,11-tetraazacyclotetra-decane-N,N′,N″,N′″-tetraacetic acid), DTTA(N¹-(p-isothiocyanatobenzyl)-diethylene-triamine-N¹,N²,N³-tetraaceticacid) and DFA(N′-[5-[[5-[[5-acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxybutanediamide).

An electrochemically active moiety is a moiety that comprises a groupthat is capable of generating an electrochemical signal in anelectrochemical method such as an amperometric or voltammetric method.Typically, an electrochemically active moiety is capable of existing inat least two distinct redox states.

A person of skill in the art would of course easily be able to select adetectable compound that would be suitable for use in accordance withthe present invention from the vast array of detectable compounds thatare routinely available. The methodology of the present invention canthus be used to produce a conjugate comprising a detectable moiety,which conjugate can then be used in any routine biochemical techniquethat involves detection of such species.

As used herein, the term “enzymatically active moiety” means an enzyme,a substrate for an enzyme or a cofactor for an enzyme. Preferably, theenzymatically active moiety is an enzyme.

As used herein, the term “affinity tag” means a chemical moiety which iscapable of interacting with an “affinity partner”, which is a secondchemical moiety, when both the affinity tag and the affinity partner arepresent in a single sample. Typically, the affinity tag is capable offorming a specific binding interaction with the affinity partner. Aspecific binding interaction is a binding interaction which is strongerthan any binding interaction that may occur between the affinity partnerand any other chemical substance present in a sample. A specific bindinginteraction may occur, for example, between an enzyme and its substrate.

Affinity tags can be useful in applications such as detection orpurification of biomolecules such as proteins. In such applications, aconjugate comprising the biomolecule and the affinity tag can bedetected or purified by exploiting the specific binding interactionbetween the affinity tag and its affinity partner.

One affinity tag/affinity partner pair that is particularly widely usedin biochemistry is the biotin/(strept)avidin pair. Avidin andstreptavidin are proteins which can be used as affinity partners forbinding with high affinity and specificity to an affinity tag derivedfrom biotin(5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoicacid). Other affinity tag/affinity partner pairs commonly used in theart include amylase/maltose binding protein,glutathione/glutathione-S-transferase and metal (for example, nickel orcobalt)/poly(His). As one of skill in the art would appreciate, eithermember of the pair could function as the “affinity tag”, with the othermember of the pair functioning as the “affinity partner”. The terms“affinity tag” and “affinity partner” are thus interchangeable.

A person of skill in the art would be aware of the routine use ofaffinity tag/affinity partner interactions in biochemistry and inparticular in the context of bioconjugate technology. A person of skillin the art would thus have no difficulty in selected an affinity tag foruse in accordance with the present invention. The methodology of thepresent invention can therefore be used to produce conjugates adaptedfor use in routine biochemical techniques that make use of affinitytag/affinity partner interactions.

Preferred affinity tags according to the present invention are biotin,amylase, glutathione and poly(His). A particularly preferred affinitytag is biotin.

As used herein, the term, the term “hapten” means a moiety whichcomprises an epitope, which is not capable of stimulating an in vivoimmune response in its native form, but which is capable of stimulatingan in vivo immune response when linked to an immunogenic carriermolecule. Typically, a hapten is a non-proteinaceous moiety ofrelatively low molecular weight (for example, a molecular weight of lessthan 1000). An epitope is the part of a molecule or moiety which isrecognized by the immune system and stimulates an immune response.

As used herein, the term “immunogenic carrier” means an antigen that isable to facilitate an immune response when administered in vivo andwhich is capable of being coupled to a hapten. Examples of immunogeniccarriers include proteins, liposomes, synthetic or natural polymericmoieties (such as dextran, agarose, polylysine and polyglutamic acidmoieties) and synthetically designed organic moieties. Commonly usedprotein immunogenic carriers have included keyhole limpet hemocyanin,bovine serum albumin, aminoethylated or cationised bovine serum albumin.thyroglobulin, ovalbumin and various toxoid proteins such as tetanustoxoid and diphtheria toxoid. Well known synthetically designed organicmolecule carriers include the multiple antigentic peptide (MAP).

As a person of skill in the biochemical art would be aware,hapten-immunogenic carrier conjugates are widely used in, for example,immunology and proteomics. A person of skill in the art would thereforerecognise that the methodology of the present invention could readily beapplied to produce conjugates comprising a hapten and an immunogeniccarrier, which conjugates could then be used in these well-establishedand routine techniques. Accordingly, in one preferred embodiment of theinvention, one of the first functional moiety and the second functionalmoiety is a hapten and the other of the first functional moiety and thesecond functional moiety is an immunogenic carrier.

As used herein, the term “antibody or antibody fragment” means a proteinthat is capable of binding to a specific antigen via an epitope on theantigen, or a fragment of such a protein. Antibodies include monoclonalantibodies and polyclonal antibodies. Monoclonal antibodies arepreferred.

As used herein, the term “antigen” means a substance that is capable ofinstigating an immune response when administered in vive and which iscapable of binding to an antibody produced during said immune response.

As used herein, the term “ligand” means a moiety that is able tointeract with a biomolecule (for example, a protein) in such a way as tomodify the functional properties of the biomolecule. Typically, theligand is a moiety that binds to a site on a target protein. Theinteraction between the ligand and the biomolecule is typicallynon-covalent. For example, the interaction may be through ionic bonding,hydrogen bonding or van der Waals' interactions. However, it is alsopossible for some ligands to form covalent bonds to biomolecules.Typically, a ligand is capable of altering the chemical conformation ofthe biomolecule when it interacts with it.

Examples of ligands capable of interacting with a protein includesubstrates (which are acted upon by the enzyme upon binding, for exampleby taking part in a chemical reaction catalysed by the enzyme),inhibitors (which inhibit protein activity on binding), activators(which increase protein activity on binding) and neurotransmitters.

As used herein, the term “biologically active moiety” means a moietythat is capable of inducing a biochemical response when administered invivo.

The biologically active moiety can be a drug. Drugs include cytotoxicagents such as doxorubicin, methotrexate and derivatives thereof,cytotoxin precursors which are capable of metabolising in vivo toproduce a cytotoxic agent, anti-neoplastic agents, anti-hypertensives,cardioprotective agents, anti-arrhythmics, ACE inhibitors,anti-inflammatories, diuretics, muscle relaxants, local anaesthetics,hormones, cholesterol lowering drugs, anti-coagulants, anti-depressants,tranquilizers, neuroleptics, analgesics such as a narcotic oranti-pyretic analgesics, anti-virals, anti-bacterials, anti-fungals,bacteriostats, CNS active agents, anti-convulsants, anxiolytics,antacids, narcotics, antibiotics, respiratory agents, anti-histamines,immunosuppressants, immunoactivating agents, nutritional additives,anti-tussives, diagnostic agents, emetics and anti-emetics,carbohydrates, glycosoaminoglycans, glycoproteins and polysaccharides,lipids, for example phosphatidyl-ethanolamine, phosphtidylserine andderivatives thereof, sphingosine, steroids, vitamins, antibiotics,including lantibiotics, bacteristatic and bactericidal agents,antifungal, anthelminthic and other agents effective against infectiveagents including unicellular pathogens, small effector molecules such asnoradrenalin, alpha adrenergic receptor ligands, dopamine receptorligands, histamine receptor ligands, GABA/benzodiazepine receptorligands, serotonin receptor ligands, leukotrienes and triodothyronine,and derivatives thereof.

The biologically active moiety can also be a moiety derived from acompound which is capable of readily crossing biological membranes andwhich, when forming a conjugate molecule with a secondary functionalmoiety, is capable of enhancing the ability of the secondary functionalmoiety to cross the biological membrane. For example, the biologicallyactive moiety may be a “protein transduction domain” (PTD) or a smallmolecule carrier (“SMC” or “molecular tug”) such as those described inWO 2009/027679, the content of which is hereby incorporated by referencein its entirety. Accordingly, in one preferred embodiment of theinvention, one of the first functional moiety and the second functionalmoiety is such a protein transduction domain or a small molecule carrierand the other of the first functional moiety and the second functionalmoiety is a drug.

In a preferred embodiment of the present invention, the biologicallyactive moiety is a drug.

As used herein, the term “liposome” means a structure composed ofphospholipid bilayers which have amphiphilic properties. Liposomessuitable for use in accordance with the present invention includeunilamellar vesicles and multilamellar vesicles.

As used herein, the term “polymeric moiety” means a single polymericchain (branched or unbranched), which is derived from a correspondingsingle polymeric molecule. Polymeric moieties may be natural polymers orsynthetic polymers. Typically, though, the polymeric molecules are notpolynucleotides.

As is well known in the biochemical field, creation of conjugatescomprising a polymeric moiety is useful in many in vivo and in vitroapplications. For example, various properties of a macromolecule such asa protein can be modified by attaching a polymeric moiety thereto,including solubility properties, surface characteristics and stabilityin solution or on freezing. Another common application involvesconjugating a polymeric moiety to a biologically active compound such asa drug with the aim of enhancing biocompatibility, reducing oreliminating immune response on administration, and/or increasing in vivostability.

A person of skill in the art would therefore recognise that themethodology of the present invention can be used to prepare a conjugatecomprising a polymeric moiety, which conjugate can then be used in anyknown application for polymeric-moiety-containing conjugates. A personof skill in the art would easily be able to select suitable polymericmoieties for use in accordance with the present invention, on the basisof those polymeric moieties used routinely in the art.

The nature of the polymeric moiety will therefore depend upon theintended use of the conjugate molecule. Exemplary polymeric moieties foruse in accordance with the present invention include polysaccharides,polyethers, polyamino acids (such as polylysine), polyvinyl alcohols,polyvinylpyrrolidinones, poly(meth)acrylic acid and derivatives thereof,polyurethanes and polyphosphazenes. Typically such polymers contain atleast ten monomeric units. Thus, for example, a polysaccharide typicallycomprises at least ten monosaccharide units.

Two particularly preferred polymeric molecules are dextran andpolyethylene glycol (“PEG”), as well as derivatives of these molecules(such as monomethoxypolyethylene glycol, “mPEG”). Preferably, the PEG orderivative thereof has a molecular weight of less than 20,000.Preferably, the dextran or derivative thereof has a molecular weight of10,000 to 500,000. In one preferred embodiment, the compounds of thepresent invention comprise a biologically active moiety, for example adrug, and a PEG or derivative thereof.

As used herein, the term “amino acid” means a moiety containing both anamine functional group and a carboxyl functional group. However,preferably the amino acid is an α-amino acid. Preferably, the amino acidis a proteinogenic amino acid, i.e. an amino acid selected from alanine,arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,glycine, histidine, isoleucine, leucine, lysine, methionine, proline,phenylalanine, pyrrolysine, selenocysteine, serine, threonine,tryptophan, tyrosine and valine. However, the amino acid can also be anon-proteinogenic amino acid. Examples of non-proteinogenic amino acidsinclude lanthionine, 2-aminoisobutyric acid, dehydroalanine,gamma-aminobutyric acid, ornithine, citrulline, canavanine and mimosine.A particularly preferred amino acid according to the present inventionis cysteine.

As used herein, the terms “peptide” and “protein” mean a polymericmoiety made up of amino acid residues. As a person of skill in the artwill be aware, the term “peptide” is typically used in the art to denotea polymer of relatively short length and the term “protein” is typicallyused in the art to denote a polymer of relatively long length. As usedherein, the convention is that a peptide comprises up to 50 amino acidresidues whereas a protein comprises more than 50 amino acids. However,it will be appreciated that this distinction is not critical since thefunctional moieties identified in the present application can typicallyrepresent either a peptide or a protein.

As used herein, the term “polypeptide” is used interchangeable with“protein”.

As used herein, a peptide or a protein can comprise any natural ornon-natural amino acids. For example, a peptide or a protein may containonly α-amino acid residues, for example corresponding to natural α-aminoacids. Alternatively the peptide or protein may additionally compriseone or more chemical modifications. For example, the chemicalmodification may correspond to a post-translation modification, which isa modification that occurs to a protein in vivo following itstranslation, such as an acylation (for example, an acetylation), analkylation (for example, a methylation), an amidation, a biotinylation,a formylation, glycosylation, a glycation, a hydroxylation, aniodination, an oxidation, a sulfation or a phosphorylation. A person ofskill in the art would of course recognise that suchpost-translationally modified peptides or proteins still constitute a“peptide” or a “protein” within the meaning of the present invention.For example, it is well established in the art that a glycoprotein (aprotein that carries one or more oligosaccharide side chains) is a typeof protein.

As used herein, the term “cell” means a single cell of a livingorganism.

As used herein, the term “carbohydrate” includes monosaccharides andoligosaccharides. Typically an oligosaccharide contains from two to ninemonosaccharide units. Thus, as used herein, a polysaccharide isclassified as a “polymeric moiety” rather than as a carbohydrate.However, a person of skill in the art will appreciate that thisdistinction is not important, since the functional moieties used inaccordance with the invention can typically constitute either of a“carbohydrate” and a “polysaccharide”.

As used herein, the term “DNA” means a deoxyribonucleic acid made up ofone or more nucleotides. The DNA may be single stranded or doublestranded. Preferably, the DNA comprises more than one nucleotide.

As used herein, the term “RNA” means a ribonucleic acid comprising oneor more nucleotides. Preferably, the RNA comprises more than onenucleotide.

As used herein, “conjugate” means a molecule which comprises a firstfunctional moiety and a second functional moiety. The first functionalmoiety and the second functional moiety are covalently linked to oneanother via a cross-linker moiety, as described herein.

As used herein, the terms “group” and “moiety” are used interchangeably.

As used herein, a “reactive group” means a functional group on a firstmolecule that is capable of taking part in a chemical reaction with afunctional group on a second molecule such that a covalent bond formsbetween the first molecule and the second molecule. Reactive groupsinclude leaving groups, nucleophilic groups, and other reactive groupsas described herein.

As used herein, the term “electrophilic leaving group” means asubstituent attached to a saturated or unsaturated carbon atom which canbe replaced by a nucleophile following a nucleophilic attack at thatcarbon atom. Those of skill in the art are routinely able to selectelectrophilic leaving groups that would be suitable for locating on aparticular compound and for reacting with a particular nucleophile.

As used herein, the term “nucleophile” means a functional group orcompound which is capable of forming a chemical bond by donating anelectron pair.

As used herein, the term “linker group” means a group which is capableof linking one chemical moiety to another. The nature of the linkergroups used in accordance with the present invention is not important. Aperson of skill in the art would recognise that linker groups areroutinely used in the construction of conjugate molecules. Typically, alinker group for use in the present invention is an organic group.Typically, such a linker group has a molecular weight of 50 to 1000,preferably 100 to 500. Examples of linker groups appropriate for use inaccordance with the present invention are common general knowledge inthe art and described in standard reference text books such as“Bioconjugate Techniques” (Greg T. Hermanson, Academic Press Inc.,1996), the content of which is herein incorporated by reference in itsentirety.

As used herein, the term “alkyl” includes both saturated straight chainand branched alkyl groups. Preferably, an alkyl group is a C₁₋₂₀ alkylgroup, more preferably a C₁₋₁₅, more preferably still a C₁₋₁₂ alkylgroup, more preferably still, a C₁₋₆ alkyl group, and most preferably aC₁₋₄ alkyl group. Particularly preferred alkyl groups include, forexample, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl,pentyl and hexyl. The term “alkylene” should be construed accordingly.

As used herein, the term “alkenyl” refers to a group containing one ormore carbon-carbon double bonds, which may be branched or unbranched.Preferably the alkenyl group is a C₂₋₂₀ alkenyl group, more preferably aC₂₋₁₅ alkenyl group, more preferably still a C₂₋₁₂ alkenyl group, orpreferably a C₂₋₆alkenyl group, and most preferably a C₂₋₄ alkenylgroup. The term “alkenylene” should be construed accordingly.

As used herein, the term “alkynyl” refers to a carbon chain containingone or more triple bonds, which may be branched or unbranched.Preferably the alkynyl group is a C₂₋₂₀ alkynyl group, more preferably aC₂₋₁₅ alkynyl group, more preferably still a C₂₋₁₂ alkynyl group, orpreferably a C₂₋₆ alkynyl group and most preferably a C₂₋₄ alkynylgroup. The term “alkynylene” should be construed accordingly.

Unless otherwise specified, an alkyl, alkenyl or alkynyl group istypically unsubstituted. However, where such a group is indicated to beunsubstituted or substituted, one or more hydrogen atoms are optionallyreplaced by halogen atoms or sulfonic acid groups. Preferably, asubstituted alkyl, alkenyl or alkynyl group has from 1 to 10substituents, more preferably 1 to 5 substituents, more preferably still1, 2 or 3 substituents and most preferably 1 or 2 substituents, forexample 1 substituent. Preferably a substituted alkyl, alkenyl oralkynyl group carries not more than 2 sulfonic acid substituents.Halogen atoms are preferred substituents. Preferably, though, an alkyl,alkenyl or alkynyl group is unsubstituted.

In the moiety that is an alkyl, alkenyl or alkynyl group or an alkylene,alkenylene or alkynylene group, in which (a) 0, 1 or 2 carbon atoms maybe replaced by groups selected from C₆₋₁₀ arylene, 5- to 10-memberedheteroarylene, C₃₋₇ carbocyclylene and 5- to 10-membered heterocyclylenegroups, and (b) 0, 1 or 2 —CH₂— groups may be replaced by groupsselected from —O—, —S—, —S—S—, —C(O)— and —N(C₁₋₆ alkyl)- groups, atotal of 0, 1 or 2 of said carbon atoms and —CH₂— groups are preferablyreplaced, more preferably a total of 0 or 1. Most preferably, none ofthe carbon atoms or —CH₂— groups is replaced.

Preferred groups for replacing a —CH₂— group are —O—, —S— and —C(O)—groups. Preferred groups for replacing a carbon atom are phenylene, 5-to 6-membered heteroarylene, C₅₋₆ carbocyclylene and 5- to 6-memberedheterocyclylene groups. As used herein, the reference to “0, 1 or 2carbon atoms” means any terminal or non-terminal carbon atom in thealkyl, alkenyl or alkynyl chain, including any hydrogen atoms attachedto that carbon atom. As used herein, the reference to “0, 1 or 2 —CH₂—groups” refers to a group which does not correspond to a terminal carbonatom in the alkyl, alkenyl or alkynyl chain.

As used herein, a C₆₋₁₀ aryl group is a monocyclic or polycyclic 6- to10-membered aromatic hydrocarbon ring system having from 6 to 10 carbonatoms. Phenyl is preferred. The term “arylene” should be construedaccordingly.

As used herein, a 5- to 10-membered heteroaryl group is a monocyclic orpolycyclic 5- to 10-membered aromatic ring system, such as a 5- or6-membered ring, containing at least one heteroatom, for example 1, 2, 3or 4 heteroatoms, selected from O, S and N. When the ring contains 4heteroatoms these are preferably all nitrogen atoms. The term“heteroarylene” should be construed accordingly.

Examples of monocyclic heteroaryl groups include thienyl, furyl,pyrrolyl, imidazolyl, thiazolyl, isothiazolyl, pyrazolyl, oxazolyl,isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, pyridinyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and tetrazolyl groups.

Examples of polycyclic heteroaryl groups include benzothienyl,benzofuryl, benzimidazolyl, benzothiazolyl, benzisothiazolyl,benzoxazolyl, benzisoxazolyl, benztriazolyl, indolyl, isoindolyl andindazolyl groups. Preferred polycyclic groups include indolyl,isoindolyl, benzimidazolyl, indazolyl, benzofuryl, benzothienyl,benzoxazolyl, benzisoxazolyl, benzothiazolyl and benzisothiazolylgroups, more preferably benzimidazolyl, benzoxazolyl and benzothiazolyl,most preferably benzothiazolyl. However, monocyclic heteroaryl groupsare preferred.

Preferably the heteroaryl group is a 5- to 6-membered heteroaryl group.Particularly preferred heteroaryl groups are thienyl, pyrrolyl,imidazolyl, thiazolyl, isothiazolyl, pyrazolyl, oxazolyl, isoxazolyl,triazolyl, pyridinyl, pyridazinyl, pyrimidinyl and pyrazinyl groups.More preferred groups are thienyl, pyridinyl, pyridazinyl, pyrimidinyl,pyrazinyl, pyrrolyl and triazinyl, most preferably pyridinyl.

As used herein, a 5- to 10-membered heterocyclyl group is anon-aromatic, saturated or unsaturated, monocyclic or polycyclic C₅₋₁₀carbocyclic ring system in which one or more, for example 1, 2, 3 or 4,of the carbon atoms are replaced with a moiety selected from N, O, S,S(O) and S(O)₂. Preferably, the 5- to 10-membered heterocyclyl group isa 5- to 6-membered ring. The term “heterocyclyene” should be construedaccordingly.

Examples of heterocyclyl groups include azetidinyl, oxetanyl, thietanyl,pyrrolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl,thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, tetrahydrothienyl,tetrahydropyranyl, tetrahydrothiopyranyl, dithiolanyl, dioxolanyl,pyrazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl,methylenedioxyphenyl, ethylenedioxyphenyl, thiomorpholinyl,S-oxo-thiomorpholinyl, S,S-dioxo-thiomorpholinyl, morpholinyl,1,3-dioxolanyl, 1,4-dioxolanyl, trioxolanyl, trithianyl, imidazolinyl,pyranyl, pyrazolinyl, thioxolanyl, thioxothiazolidinyl,1H-pyrazol-5-(4H)-onyl, 1,3,4-thiadiazol-2(3H)-thionyl, oxopyrrolidinyl,oxothiazolidinyl, oxopyrazolidinyl, succinimido and maleimido groups andmoieties. Preferred hetenrocyclyl groups are pyrrolidinyl,imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl,isothiazolidinyl, tetrahydrofuranyl, tetrahydrothienyl,tetrahydropyranyl, tetrahydrothiopyranyl, dithiolanyl, dioxolanyl,pyrazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl,thiomorpholinyl and morpholinyl groups and moieties. More preferredheterocyclyl groups are tetrahydropyranyl, tetrahydrothiopyranyl,thiomorpholinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl,morpholinyl and pyrrolidinyl groups.

For the avoidance of doubt, although the above definitions of heteroaryland heterocyclyl groups refer to an “N” moiety which can be present inthe ring, as will be evident to a skilled chemist the N atom will beprotonated (or will carry a substituent as defined below) if it isattached to each of the adjacent ring atoms via a single bond.

As used herein, a C₃₋₇ carbocyclyl group is a non-aromatic saturated orunsaturated hydrocarbon ring having from 3 to 7 carbon atoms. Preferablyit is a saturated or mono-unsaturated hydrocarbon ring (i.e. acycloalkyl moiety or a cycloalkenyl moiety) having from 3 to 7 carbonatoms, more preferably having from 5 to 6 carbon atoms. Examples includecyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and theirmono-unsaturated variants. Particularly preferred carbocyclic groups arecyclopentyl and cyclohexyl. The term “carbocyclylene” should beconstrued accordingly.

Where specified, 0, 1 or 2 carbon atoms in a carbocyclyl or heterocyclylgroup may be replaced by —C(O)— groups. As used herein, the “carbonatoms” being replaced are understood to include the hydrogen atoms towhich they are attached. When 1 or 2 carbon atoms are replaced,preferably two such carbon atoms are replaced. Preferred suchcarbocyclyl groups include a benzoquinone group and preferred suchheterocyclyl groups include succinimido and maleimido groups.

Unless otherwise specified, an aryl, heteroaryl, carbocyclyl orheterocyclyl group is typically unsubstituted. However, where such agroup is indicated to be unsubstituted or substituted, one or morehydrogen atoms are optionally replaced by halogen atoms or C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ alkylthiol, —N(C₁₋₆ alkyl)(C₁₋₆ alkyl), nitro orsulfonic acid groups. Preferably, a substituted aryl, heteroaryl,carbocyclyl or heterocyclyl group has from 1 to 4 substituents, morepreferably 1 to 2 substituents and most preferably 1 substituent.Preferably a substituted aryl, heteroaryl, carbocyclyl or heterocyclylgroup carries not more than 2 nitro substituents and not more than 2sulfonic acid substituents. Preferred substituents are halogen atoms andC₁₋₄ alkyl and C₁₋₄ alkoxy groups. Particularly preferred substituentsare halogen atoms. Preferably, though, an aryl, heteroaryl, carbocyclylor heterocyclyl group is unsubstituted.

As used herein, halogen atoms are typically F, Cl, Br or I atoms,preferably Br or Cl atoms, more preferably Br atoms.

As used herein, a C₁₋₆ alkoxy group is a C₁₋₆ alkyl (e.g. a C₁₋₄alkyl)group which is attached to an oxygen atom.

As used herein, a C₁₋₆ alkylthiol group is a C₁₋₆ alkyl (e.g. a C₁₋₄alkyl) group which is attached to a sulfur atom.

As used herein, a 5- to 10-membered heterocyclylthiol is a 5- to10-membered (e.g., a 5- to 6-membered) heterocyclyl group which isattached to a sulfur atom.

As used herein, a C₆₋₁₀ arylthiol is a C₆₋₁₀ aryl (e.g., a phenyl) groupwhich is attached to a sulfur atom.

As used herein, a C₃₋₇ carbocyclylthiol is a C₃₋₇ carbocyclyl (e.g., aC₅₋₆ carbocyclyl) group which is attached to a sulfur atom.

In the present invention the moiety of formula (I) constitutes across-linking reactive moiety which is capable of linking together athiol-containing first functional moiety and a second functional moiety.

Preferably X and X′ are the same or different and each representsoxygen, sulfur or a group of formula ═NH. More preferably, X and X′ arethe same or different and each represents oxygen or sulfur. Preferablyat least one of X and X′ represents oxygen. Most preferably, X and X′are both oxygen.

Y is preferably a halogen atom or a triflate, tosylate, mesylate,N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, C₁₋₆ alkylthiol, 5-to 10-membered heterocyclylthiol, C₆₋₁₀ arylthiol, C₃₋₇carbocyclylthiol, —OC(O)CH₃, —OC(O)CF₃, phenyloxy, —NR_(x)R_(y)R_(z) ⁺or —PR_(x)R_(y)R_(z) ⁺ group. More preferably, Y is a halogen atom or atriflate, tosylate, mesylate, N-hydroxysuccinimidyl,N-hydroxysulfosuccinimidyl. C₁₋₆ alkylthiol, 5- to 10-memberedheterocyclylthiol, (C₆₋₁₀ arylthiol or C₃₋₇ carbocyclylthiol. Morepreferably still Y is a halogen atom or a C₁₋₆ alkylthiol, 5- to10-membered heterocyclylthiol, C₆₋₁₀ arylthiol or C₃₋₇ carbocyclylthiolgroup. Most preferably Y is a halogen atom, particularly a bromine atom.

R_(x), R_(y) and R_(z) are each preferably selected from hydrogen atomsand C₁₋₆ alkyl groups. More preferably R_(x), R_(y) and R_(z) are eachpreferably selected from hydrogen atoms and methyl and ethyl groups.Preferably, in a particular —NR_(x)R_(y)R_(z) ⁺ or —PR_(x)R_(y)R_(z) ⁺group. R_(x), R_(y) and R_(z) are the same.

The compound of formula R₁—H comprises at least a first thiol group, SH.In the present invention, this first thiol group is capable of reactingwith the moiety of formula (I) by nucleophilic attack at the 2-position.The outcome of reacting the compound of formula R—H with the moiety offormula (I) is a moiety in which the electrophilic leaving group offormula Y at the 2-position in the moiety of formula (I) is replaced bythe group of formula R₁. More specifically, the group of formula R₁becomes attached at the 2-position through a thiol bond of formula —S—which is derived from the first SH group on the corresponding compoundof formula R₁—H. It will therefore be clear that the hydrogen atom inthe first thiol group SH of R₁—H constitutes the hydrogen atom attachedto the group of formula R₁. Thus, when R₁ becomes attached to thecross-linker the hydrogen atom in this first thiol group is lost inorder to form the —S— bond between R₁ and the cross-linker.

R₁ can be a group of formula —S-L-F₁, in which case the sulfur atom ofthe first thiol group is attached to the linker group of formula L. Inthis embodiment, therefore, it will be clear that the linker can be usedto provide a thiol group capable of reacting with the moiety of formula(I), which linker group is then attached to a first functional moietythat does not contain such a thiol group. However, preferably R₁ is agroup of formula F₁, and more particularly a first functional moietywhich, together with the H-atom to which it is attached in the compoundof formula R₁—H, contains a first SH group.

In a preferred embodiment, at least one of the first functional moietyand the second functional moiety is an enzymatically active moiety, ahapten, an immunogenic carrier, an antibody or antibody fragment, anantigen, a ligand, a biologically active moiety, an amino acid, apeptide, a protein, a carbohydrate, a DNA or an RNA. Preferably thefirst functional moiety is an enzymatically active moiety, a hapten, animmunogenic carrier, an antibody or antibody fragment, an antigen, aligand, a biologically active moiety, an amino acid, a peptide, aprotein, a carbohydrate, a DNA or an RNA. Preferred combinations offirst functional moiety/second functional moiety include those set outin Table 1 below.

TABLE 1 Preferred combinations of first functional moiety/secondfunctional moiety First or second functional moiety Second or firstfunctional moiety Antibody or antibody fragment, amino Detectable moietyacid, peptide, protein, DNA or RNA Hapten Immunogenic carrier Antibodyor antibody fragment Enzymatically active moiety (preferably an enzyme)Antibody or antibody fragment Biologically active moiety, for example acytotoxic agent or cytoxin precursor Detectable moiety, enzymaticallyactive Liposome moiety, antibody or antibody fragment, antigen, hapten,biologically active moiety, amino acid, peptide, protein, DNA or RNAEnzymatically active moiety, antibody Affinity tag or antibody fragment,antigen, amino acid, peptide, protein, DNA or RNA Biologically activemoiety Polymeric moiety Enzymatically active moiety DNA or RNA

In a particularly preferred embodiment, R₁ is a group of formula F₁ andF₁ is a peptide or protein comprising at least a first cysteine residue.For the avoidance of doubt, a cysteine residue in a peptide or proteinis a residue of formula

wherein in the compound of formula R₁—H the peptide or protein isattached to the hydrogen atom through the sulfur atom of the cysteineresidue. In this embodiment, it will be understood that the “firstcysteine residue” means a cysteine residue that is located at such aposition on the peptide or protein such that it can react with themoiety of formula (I). More specifically, the group R₁ becomes attachedto the moiety of formula (I) via nucleophilic attack of the thiol groupof the first cysteine residue at the 2-position of the moiety of formula(I), such that the group Y is replaced by the thiol group in the firstcysteine residue in the group R₁.

In a particularly preferred embodiment of the invention where R₁ is agroup of formula F₁ and F₁ is a peptide or protein comprising at least afirst cysteine residue, R₁ further comprises at least a second cysteineresidue. For the avoidance of doubt, the second cysteine residue islocated at such a position on the peptide or protein such that it canalso react with the moiety of formula (I). Furthermore, in thisembodiment the moiety of formula (I) is a compound of formula (Ia)wherein the group R₂ is an electrophilic leaving group of formula Y. Thegroup R₁ then becomes further attached to the compound of formula (Ia)via nucleophilic attack of the thiol group of the second cysteineresidue at the 3-position of the moiety of formula (Ia), such that thegroup R₂ is replaced by the thiol group in the second cysteine residuein the group R₁. This embodiment of the invention is particularly usefulwhen the first functional moiety is a peptide or protein containing adisulfide bridge, since it allows the cross-linker reagent to be addedacross the disulfide bridge. Preferably, when a peptide or proteincontaining a disulfide bridge is to be reacted with the moiety offormula (I), the disulfide bridge is first reduced using techniquesknown in the art. For example, the reduction can be carried out by usingstandard phosphine reagents such as (tris(2-carboxyethyl)phosphine) orby carrying out a thiol-disulfide exchange reaction.

Reduction of a disulfide group can be carried out by reaction with areducing agent such as a phosphine, a thiol or a hydride agent.Preferred reducing agents are tris(2-carboxyethyl)phosphine,glutathione, 2-mercaptoethanol and dithiothreitol. A preferred group ofreagents is 1,2-ethanedithiol, 2-mercaptoethanol, dithiothreitol,glutathione and tris(2-carboxyethyl)phosphine.

It will be clear that the moiety of formula (I) represents the keyreactive moiety which according to the present invention allows athiol-containing functional moiety to be conjugated to a secondaryfunctional moiety. Accordingly, a compound containing the moiety offormula (I) can be used as reagent for linking a first functional moietyto a second functional moiety. In the moiety of formula (I) (and themoiety of formula (II), as described in detail elsewhere), the symbol

-   -           means a point of attachment to another group. It will be        appreciated that the identity of the groups attached via these        points of attachment is unimportant to the present invention.        Those of skill in the art would readily understand that it would        be possible to choose the groups attached via these points of        attachment to suit a particular purpose, for example based on        the specific identity of the functional moieties to be linked        together. As would be well known to those of skill in the art,        cross-linking reagents are routinely designed which carry        functional groups adapted to react with functional moieties        having particular reactive groups and which are spaced by linker        groups (which typically do not play a significant role in the        reactions). A person of skill in the art would immediately        understand that the moiety of formula (I) could readily be        incorporated into routine cross-linker reagents, for example, by        replacing conventional moieties designed to react with thiol        groups (for example, maleimide groups). Detailed information on        the design of cross-linker reagents suitable for adaptation in        this manner can be found, for example, in “Bioconjugate        Techniques” (Greg T. Hermanson, Academic Press Inc., 1996), the        content of which is herein incorporated by reference in its        entirety.

The moiety of formula (I) is capable of linking together at least afirst functional moiety and a second functional moiety. Where across-linker compound containing a moiety of formula (I) does not carryany reactive groups other than those on the moiety of formula (I), thefirst functional moiety can react at the 2-position by replacing theleaving group Y and the second functional moiety can then add byelectrophilic addition across the carbon-carbon double bond between the2- and 3-positions. Alternatively, the cross-linker reagent may compriseone or more additional reactive groups capable of reacting with furtherfunctional moieties.

In a further embodiment, the second functional moiety may be a moietycontaining an alkene moiety and can be attached to the moiety of formula(I) by engaging in a photocatalytic [2+2] cycloaddition with thecarbon-carbon double bond between the 2- and 3-positions of the moietyof formula (I). This procedure results in a cyclobutane ring moietycontaining the 2- and 3-carbon atoms from the moiety of formula (I) andin which the carbon-carbon double bond has been saturated.

Preferably, the compound containing a moiety of formula (I) according tothe present invention is a compound of formula (Ia)

wherein:

-   -   X and X′ are the same or different and each represents oxygen,        sulfur or a group of formula ═NQ, in which Q is hydrogen,        hydroxyl, C₁₋₆ alkyl or phenyl;    -   either:        -   R₃ and R₃′ are the same or different and each represents a            hydrogen atom or a group of formula E, Nu, -L(Z)_(n) or IG;            or        -   R₃ and R₃′ together form a group of formula —O— or            —N(R_(33′)), wherein R_(33′) represents a hydrogen atom or a            group of formula Y, Nu, -L(Z)_(n) or IG; or        -   R₃ and R₃′ together form a group of formula            —N(R_(33′))—N(R_(33′))—, wherein each R_(33′) is the same or            different and represents a hydrogen atom or a group of            formula Y, Nu, -L(Z)_(n) or IG;    -   R₂ represents a hydrogen atom or a group of formula Y, Nu,        -L(Z)_(n) or IG;    -   each group of formula E and Y is the same or different and        represents an electrophilic leaving group;    -   each group of formula Nu is the same or different and represents        a nucleophile selected from —OH, —SH, —NH₂ and —NH(C₁₋₆ alkyl);    -   each group of formula I, is the same or different and represents        a linker group;    -   each group of formula Z is the same or different and represents        a reactive group attached to a group of formula L which is        capable of reacting with a compound containing a second        functional moiety as defined in claim 1 such that said second        functional moiety becomes linked to said group of formula L;    -   n is 1, 2 or 3; and    -   each group of formula IG is the same or different and represents        a moiety which is a C₁₋₂₀ alkyl group, a C₂₋₂₀ alkenyl group or        a C₂₋₂₀ alkynyl group, which is unsubstituted or substituted by        one or more substituents selected from halogen atoms and        sulfonic acid groups, and in which (a) 0, 1 or 2 carbon atoms        are replaced by groups selected from C₆₋₁₀ arylene, 5- to        10-membered heteroarylene, C₃₋₇ carbocyclylene and 5- to        10-membered heterocyclylene groups, and (b) 0, 1 or 2 —CH₂—        groups are replaced by groups selected from —O—, —S—, —S—S—,        —C(O)— and —N(C₁₋₆ alkyl)- groups, wherein:    -   (i) said arylene, heteroarylene, carbocyclylene and        heterocyclylene groups are unsubstituted or substituted by one        or more substituents selected from halogen atoms and C₁₋₆ alkyl,        C₁₋₆ alkoxy, C₁₋₆ alkylthiol, —N(C₁₋₆ alkyl)(C₁₋₆ alkyl), nitro        and sulfonic acid groups; and    -   (ii) 0, 1 or 2 carbon atoms in said carbocyclylene and        heterocyclylene groups are replaced by —C(O)— groups.

Preferably in the compound of formula (Ia) R₃ and R₃′ are the same ordifferent and each represents a hydrogen atom or a group of formula E,Nu, -L(Z)_(n) or IG; or R₃ and R₃′ together form a group of formula—N(R_(33′)), wherein R_(33′) represents a hydrogen atom or a group offormula Y, Nu, -L(Z)_(n) or IG.

Preferred groups X, X′ and Y in the formula (Ia) are as defined above.

When R₃ and R₃′ are the same or different and each represents a hydrogenatom or a group of formula E, Nu, -L(Z)_(n) or IG, preferably R₃ and R₃′are the same or different and each represents a group of formula E, Nu,-L(Z)_(n) or IG. In this embodiment, preferably at least one of R₃ andR₃′ represents a group of formula E, Nu or -L(Z)_(n).

Preferably R₃ and R₃′ together form a group of formula —N(R_(33′)).

R_(33′) preferably represents a hydrogen atom or a group of formula-L(Z)_(n) or IG. Particularly preferred R_(33′) groups are hydrogenatoms and groups of formula IG. Most preferably, R_(33′) is a hydrogenatom or a C₁₋₆ alkyl group.

R₂ is preferably a hydrogen atom or a group of formula Y, -L(Z)_(n) orIG. More preferably, R₂ is preferably a hydrogen atom or a group offormula Y or IG. Most preferably, R₂ is a hydrogen or halogen atom or aC₁₋₆ alkyl group.

E is preferably a halogen atom or a C₁₋₆ alkoxy, thiol, C₁₋₆ alkylthiol,—N(C₁₋₆ alkyl)(C₁₋₆ alkyl), triflate, tosylate, mesylate,N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, imidazolyl, phenyloxyor nitrophenyloxy group. More preferred groups of formula E are halogenatoms and triflate, tosylate and mesylate groups.

Nu is preferably a group of formula —OH or —SH. In another embodiment,Nu is preferably a group of formula —OH, —NH₂ or —SH, more preferably—NH₂ or —SH.

The linker moiety L links together two other moieties in the compoundsof the present invention (i.e., it is at least a divalent moiety).However, in some embodiments certain linker moieties L may link togethermore than two other moieties (for example, where R₂, R₃, R₃′ or R_(33′)represents -L(Z)_(n) wherein n is 2 or 3), in which case it is to beunderstood that the third other moiety and any further other moiety eachreplace a hydrogen atom on the corresponding divalent linker moiety L.

L preferably represents a moiety which is a C₁₋₂₀ alkylene group, aC₂₋₂₀ alkenylene group or a C₂₋₂₀ alkynylene group, which isunsubstituted or substituted by one or more substituents selected fromhalogen atoms and sulfonic acid groups, and in which (a) 0, 1 or 2carbon atoms are replaced by groups selected from C₆₋₁₀ arylene, 5- to10-membered heteroarylene, C₃₋₇ carbocyclylene and 5- to 10-memberedheterocyclylene groups, and (b) 0, 1 or 2 —CH₂— groups are replaced bygroups selected from —O—, —S—, —S—S—, —C(O)— and —N(C₁₋₆ alkyl)- groups,wherein:

-   (i) said arylene, heteroarylene, carbocyclylene and heterocyclylene    groups are unsubstituted or substituted by one or more substituents    selected from halogen atoms and C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆    alkylthiol, —N(C₁₋₆ alkyl)(C₁₋₆ alkyl), nitro and sulfonic acid    groups; and-   (ii) 0, 1 or 2 carbon atoms in said carboxcyclylene and    heterocyclylene groups are replaced by —C(O)— groups.

More preferably, L represents a moiety which is an unsubstituted C₁₋₆alkylene group, C₂₋₆ alkenylene group or C₂₋₆alkynylene group, in which(a) 0 or 1 carbon atom is replaced by a group selected from phenylene,5- to 6-membered heteroarylene, C₅₋₆ carbocyclylene and 5- to 6-memberedheterocyclylene groups, wherein said phenylene, heteroarylene,carbocyclylene and heterocyclylene groups are unsubstituted orsubstituted by one or two substituents selected from halogen atoms andC₁₋₄ alkyl and C₁₋₄ alkoxy groups, and (b) 0, 1 or 2 —CH₂— groups arereplaced by groups selected from —O—, —S— and —C(O)— groups.

Most preferably, L is a moiety which is an unsubstituted C₁₋₄alkylenegroup, in which 0 or 1 carbon atom is replaced by an unsubstitutedphenylene group.

Z represents a reactive group attached to a group of formula L which iscapable of reacting with a functional moiety such that the functionalmoiety becomes linked to the group of formula L. As those of skill inthe art would understand, the nature of the reactive group itself is notimportant. A very wide range of reactive groups are now routinely usedin the art to connect a functional moiety to a cross-linker reagent.Such reactive groups may be capable, for example, of attaching an aminecompound, a thiol compound, a carboxyl compound, a hydroxyl compound, acarbonyl compound or a compound containing a reactive hydrogen, to across-linker. Those of skill in the art would of course immediatelyrecognise that any such reactive group would be suitable for use inaccordance with the present invention. Those of skill in the art wouldbe able to select an appropriate reactive group from common generalknowledge, with reference to standard text books such as “BioconjugateTechniques” (Greg T. Hermanson, Academic Press Inc., 1996), the contentof which is herein incorporated by reference in its entirety.

Z is preferably:

-   (a) a group of formula -LG, —C(O)-LG, —C(S)-LG or —C(NH)-LG wherein    LG is an electrophilic leaving group;-   (b) a nucleophile Nu′ selected from —OH, —SH, —NH₂, —NH(C₁₋₆ alkyl)    and —C(O)NHNH₂ groups;-   (c) a cyclic moiety Cyc, which is capable of a ring-opening    electrophilic reaction with a nucleophile;-   (d) a group of formula —S(O₂)(Hal), wherein Hal is a halogen atom;-   (e) a group of formula —N═C═O or —N═C═S;-   (f) a group of formula —S—S(IG′) wherein IG′ represents a group of    formula IG as defined herein;-   (g) a group AH, which is a C₆₋₁₀ aryl group that is substituted by    one or more halogen atoms;-   (h) a photoreactive group capable of being activated by exposure to    ultraviolet light;-   (i) a group of formula —C(O)H or —C(O)(C₁₋₆ alkyl);-   (j) a maleimido group;-   (k) a group of formula —C(O)CHCH₂;-   (l) a group of formula —C(O)C(N₂)H or -PhN₂ ⁺, where Ph represents a    phenyl group; or-   (m) an epoxide group.

Most preferably, Z is selected from:

-   (a) groups of formula -LG, —C(O)-LG and —C(S)-LG, wherein LG is    selected from halogen atoms and —O(C₁₋₆ alkyl), —SH, —S(C₁₋₆ alkyl),    triflate, tosylate, mesylate, N-hydroxysuccinimidyl and    N-hydroxysulfosuccinimidyl groups;-   (b) groups of formula —OH, —SH and —NH₂;-   (c) a group of formula

and

-   (d) a maleimido group.

LG is preferably selected from halogen atoms and —O(IG′), —SH, —S(IG′),—NH₂, NH(IG′), —N(IG)(IG′), —N₃, triflate, tosylate, mesylate,N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, imidazolyl and azidegroups, wherein IG′ and IG″ are the same or different and eachrepresents a group of formula IG.

Nu′ is preferably selected from —OH, —SH and —NH₂ groups.

-   Cyc is preferably selected from the groups

Hal is preferably a chlorine atom.

AH is preferably a phenyl group that is substituted by at least onefluorine atom.

The photoreactive group is preferably selected from:

-   (a) a C₆₋₁₀ aryl group which is substituted by at least one group of    formula —N₃ and which is optionally further substituted by one or    more halogen atoms;-   (b) a benzophenone group;-   (c) a group of formula —C(O)C(N₂)CF₃; and-   (d) a group of formula -PhC(N₂)CF₃, wherein Ph represents a phenyl    group.

n is preferably 1 or 2, and most preferably 1.

IG preferably represents a moiety which is an unsubstituted C₁₋₆ alkylgroup, C₂₋₆ alkenyl group or C₂₋₆ alkynyl group, in which (a) 0 or 1carbon atom is replaced by a group selected from phenylene, 5- to6-membered heteroarylene, C₅₋₆ carbocyclylene and 5- to 6-memberedheterocyclylene groups, wherein said phenylene, heteroarylene,carbocyclylene and heterocyclylene groups are unsubstituted orsubstituted by one or two substituents selected from halogen atoms andC₁₋₄ alkyl and C₁₋₄ alkoxy groups, and (b) 0, 1 or 2 —CH₂— groups arereplaced by groups selected from —O—, —S— and —C(O)— groups.

More preferably, IG represents a moiety which is an unsubstituted C₁₋₆alkyl group, in which (a) 0 or 1 carbon atom is replaced by a groupselected from unsubstituted phenylene, 5- to 6-membered heteroarylene,C₅₋₆ carbocyclylene and 5- to 6-membered heterocyclylene groups.

Most preferably, IG represents an unsubstituted C₁₋₆ alkyl group.

Preferably the compound of formula (Ia) is a compound of formula (Ib):

wherein X, X′, Y, R₂ and R_(33′) are all as herein defined.

In the compound of formula (Ib), preferably:

-   -   X and X′ each represent an oxygen atom;    -   R_(33′) represents a hydrogen atom or a C₁₋₆ alkyl group;    -   Y represents a halogen atom; and    -   R₂ represents a hydrogen or halogen atom or a C₁₋₆ alkyl group.

Embodiment (1) of the present invention relates to use of a compoundcontaining the moiety of formula (I), as defined above, as a reagent forlinking the compound of formula R₁—H which comprises a first functionalmoiety of formula F₁ to a second functional moiety of formula F₂.Typically, this use involves carrying out a process of the presentinvention, as defined in embodiment (2) or embodiment (3) describedbelow.

Preferably in the use of embodiment (1) the compound containing a moietyof formula (I) is not dibromomaleic acid if the compound of formula R₁—His 2-mercaptoethanol. Embodiment (2) of the present invention relates toa process for producing a conjugate. In a step (i) of this process, acompound containing a moiety of formula (I) is reacted with a compoundof formula R₁—H to produce a compound containing a moiety of formula(II):

This step (i) involves attaching the group R₁ via nucleophilic attack ofthe first SH group in the compound of formula R₁—H at the 2-position ofthe moiety of formula (I), such that the group Y at the 2-position isreplaced by the group R₁.

In a step (ii) of the process of embodiment (2), a moiety of formula F₂is linked to the moiety of formula (II), thus producing the conjugate.Several procedures can be used to carry out step (ii), for example thoseset out under paragraphs (a), (b), (c) and (d) below:

-   (a) The process may comprise linking F₂ to the moiety of    formula (II) via an electrophilic addition reaction of F₂ across the    carbon-carbon double bond between the 2-position and the 3-position    of the formula (II).-   (b) Where the compound containing a moiety of formula (I) is a    compound of formula (Ia), R₃ and R₃′ together form a group of    formula —N(R_(33′)), and R_(33′) represents a hydrogen atom or a    group of formula Y, Nu or -L(Z)_(n), the process may comprise    linking F₂ to the moiety of formula (II) via a reaction between F₂    and (i) the nitrogen atom of the moiety of formula —N(R_(33′))    or (ii) said group of formula Y, Nu or -L(Z)_(n).-   (c) Where the compound containing a moiety of formula (I) is a    compound of formula (Ia), R₃ and R₃′ do not together a group of    formula —N(R_(33′)), and at least one of R₃ and R₃′ represents a    group of formula E, Nu or -L(Z)_(n), the process may comprise    linking F₂ to the moiety of formula (II) via a reaction between F₂    and said group of formula E, Nu or -L(Z)_(n).-   (d) Where the compound containing a moiety of formula (I) is a    compound of formula (Ia) and R₂ represents a group of formula Y, Nu    or -L(Z)_(n), the process may comprise linking F₂ to the moiety of    formula (II) via a reaction between F₂ and said group of formula Y,    Nu or -L(Z)_(n).

In a further embodiment, the moiety of formula F₂ is linked to themoiety of formula (II) by a effecting a photocatalytic [2+2]cycloaddition reaction between an alkene group on the moiety of formulaF₂ and the carbon-carbon double bond between the 2-position and the3-position of the formula (II). This procedure results in a cyclobutanering moiety containing the 2- and 3-carbon atoms from the moiety offormula (II) and in which the carbon-carbon double bond has beensaturated.

In a still further embodiment, when R₃ and R₃′ together form a group offormula —O— and the moiety of formula F₂ carries a nucleophilic group,such as a primary or secondary amine group, the moiety of formula F₂ canlink to the moiety of formula (II) by engaging in a nucleophilicring-opening and then nucleophilic ring closing reaction. For example,when X and/or X′ are O and R₃ and R₃′ together form a group of formula—O—, the moiety of formula (II) is a cyclic acid anhydride. Thus, it canbe seen that a moiety of formula F₂ carrying, for example, an aminegroup can engage in nucleophilic ring-opening and then nucleophilic ringclosing with the overall effect that the group —O— is replaced by thegroup —N(functional moiety)-.

An alternative process for producing a conjugate is provided byembodiment (3) of the present invention. In this process, a compound offormula R₁—H is reacted with a compound comprising (a) a moiety offormula (I) and (b) at least one moiety of formula F₂ linked thereto.The process involves attaching the group R₁ via nucleophilic attack ofthe first SH group in the compound of formula R₁—H at the 2-position ofthe moiety of formula (I), such that the group Y at the 2-position isreplaced by the group R₁.

Preferably according to the above-described process of embodiment (3),the compound comprising (a) the moiety of formula (I) and (b) at leastone moiety of formula F₂ linked thereto, is a compound of formula (IIa)

wherein:

-   -   either:        -   R_(3a) represents a group of formula R₃ or a group of            formula F₂ or -L(F₂)_(m)(Z)_(n-m) and R_(3a)′ independently            represents a group of formula R₃′ or a group of formula F₂            or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula —O— or            —N(R_(33a′)), wherein R_(33a′) represents a group of formula            R_(33′) or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula            —N(R_(33a′))—N(R_(33a′))—, wherein each R_(33a′) is the same            or different and represents a group of formula R_(33′) or a            group of formula F₂ or -L(F₂)_(m)(Z)_(n-m);    -   R_(2a) represents a group of formula R₂ or a group of formula F₂        or -L(F₂)_(m)(Z)_(n-m);    -   m is an integer having a value of from zero to n;    -   the compound of formula (IIa) comprises at least one group of        formula F₂; and    -   F₂, X, X′, R₃, R₃′, R_(33′), R₂, L, Z and n are all as defined        herein.

Preferably according to the above-described process of embodiment (3),R_(3a) represents a group of formula R₃ or a group of formula F₂ or-L(F₂)_(m)(Z)_(n-m), and R_(3a)′ independently represents a group offormula R₃′ or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m); or R_(3a)and R_(3a)′ together form a group of formula —N(R_(33a′)), whereinR_(33a′) represents a group of formula R_(33′) or a group of formula F₂or -L(F₂)_(m)(Z)_(n-m).

As will be clear to those of skill in the art, the compound of formula(IIa) is related to the compound of formula (II) as described above.However, the compound of formula (IIa) comprises at least one functionalmoiety F₂. Accordingly, the compound of formula (IIa) can readily beprepared by linking a functional moiety F₂ to its corresponding compoundof formula (II) using methods routinely known in the art.

Preferably according to the above-described process of embodiment (3),the compound comprising (a) the moiety of formula (I) and (b) at leastone moiety of formula F₂ linked thereto, is a compound of formula (IIa)in which:

-   -   either:        -   R_(3a) represents a group of formula R₃ or a group of            formula F₂ and R_(3a)′ independently represents a group of            formula R₃′ or a group of formula F₂; or        -   R_(3a) and R_(3a)′ together form a group of formula            —N(R_(33a′)), wherein R_(33a′) represents a group of formula            R_(33′) or a group of formula F₂; and    -   R_(2a) represents a group of formula R₂ or a group of formula        F₂.

Preferably the compound of formula (IIa) comprises at most three groupsof formula F₂, more preferably one or two groups of formula F₂, and mostpreferably one group of formula F₂.

Clearly, after carrying the process of embodiment (2) or (3) of thepresent invention one or more further reactive groups may remain on theconjugate product (including a carbon-carbon double bond located at aposition corresponding to the 2- and 3-positions of the cross-linkerreagent as well as further nucleophilic groups, electrophilic groups,and reactive groups of formula Z). Accordingly, in further aspects theprocesses of embodiments (2) and (3) of the present invention furthercomprise linking one or more further functional moieties to saidconjugate, wherein each further functional moiety is the same ordifferent and is selected from a detectable moiety, an enzymaticallyactive moiety, an affinity tag, a hapten, an immunogenic carrier, anantibody or antibody fragment, an antigen, a ligand, a biologicallyactive moiety, a liposome, a polymeric moiety, an amino acid, a peptide,a protein, a cell, a carbohydrate, a DNA and an RNA.

The chemical reactions taking place in the processes of embodiments (2)and (3) can be carried out using routine techniques known in the art forattaching cross-linker reagents to functional moieties, such as thosedescribed in “Bioconjugate Techniques” (Greg T. Hermanson, AcademicPress Inc., 1996), the content of which is herein incorporated byreference in its entirety. Further examples of suitable conditions forcarrying out such reactions can be found in the Examples section of thepresent specification.

As would be understood by those of skill in the art, where a reagent(for example, a compound carrying a functional moiety or a cross-linkerreagent) carries more than one reactive group, it may be desirable toeffect chemical protection of reactive groups that are not intended totake part in the reaction. For example, it may be necessary to protectgroups such as hydroxyl, amino and carboxy groups, where these aredesired in the final product, to avoid their unwanted participation inthe reactions (see, for example, Greene, T. W., “Protecting Groups inOrganic Synthesis”. John Wiley and Sons, 1999). Conventional protectinggroups may be used in conjunction with standard practice. In someinstances deprotection may be used in an intermediate or final step, andthus the processes of embodiments (2) and (3) according to the inventiondescribed herein are understood to extend to addition and removal ofsuch protecting groups.

Preferably in embodiment (2) the compound containing a moiety of formula(I) is not dibromomaleic acid if the compound of formula R₁—H is2-mercaptoethanol.

Preferably in embodiment (3) the compound comprising (a) a moiety offormula (I) and (b) at least one moiety of formula F₂ linked thereto isnot N-phenyl 3,4-dibromomaleimide, wherein the N-phenyl groups issubstituted or unsubstituted, if the compound of formula R₁—H is2-mercaptoethanol.

It will be appreciated that in some embodiments the conjugate producedaccording to the process of embodiment (2) or (3) will contain amaleimide ring. Specifically, this occurs when in the moiety of formula(I) the carbon atoms at positions 1 and 4 are linked together via agroup —N(R_(33′))—. When the conjugate comprises a maleimide ring theprocess of embodiment (2) or (3) may further comprise effecting ringopening of said maleimide ring. Ring opening of maleimide rings can beeffected by hydrolysis reactions that are known in the art. Effectingring opening of the maleimide may be advantageous in certainapplications since it can render the functional moieties irreversiblybound to the conjugate.

In embodiment (4), the present invention relates to a process forcleaving the bond between a thiol-containing functional moiety and thecross-linker moiety (which may additionally be linked to one or morefurther functional moieties). More specifically, the cleavage iseffected on a compound comprising a moiety of formula (II).

Examples of techniques in which the process of embodiment (4) isparticularly usefully include protein purification, proteomic analysisand processes for probing the binding site of an enzyme. In a preferredembodiment of the process of embodiment (4) the first functional moietyof formula F₁ is a protein, especially a protein that is expensive ortime-consuming to obtain, such as proteins that are difficult to express(e.g., a GPCR protein). In another preferred embodiment of the processof embodiment (4), the first functional moiety of formula F₁ is abiologically active moiety (e.g., a drug) since here the methodology canbe exploited in, for example, drug delivery methods.

In a first preferred embodiment of the process of embodiment (4) thecompound comprising a moiety of formula (II) is a compound of formula(III)

wherein:

-   -   either:        -   R_(3a) represents a group of formula R₃ or a group of            formula F₂ or -L(F₂)_(m)(Z)_(n-m) and R_(3a)′ independently            represents a group of formula R₃′ or a group of formula F₂            or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula —O— or            —N(R_(33a′)), wherein R_(33a′) represents a group of formula            R_(33′) or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m); or        -   R₃, and R_(3a)′ together form a group of formula            —N(R_(33a′))—N(R_(33a′))—, wherein each R_(33a′) is the same            or different and represents a group of formula R_(33a′) or a            group of formula F₂ or -L(F₂)_(m)(Z)_(n-m);    -   R_(2a) represents a group of formula R₂ or a group of formula F₂        or -L(F₂)_(m)(Z)_(n-m);    -   m is an integer having a value of from zero to n; and    -   R₁, F₂, X, X′, R₃, R₃′, R_(33′), R₂, L, Z and n are all as        defined herein.

More preferably in this embodiment, R_(3a) represents a group of formulaR₃ or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m) and R_(3a)′independently represents a group of formula R₃′ or a group of formula F₂or -L(F₂)_(m)(Z)_(n-m); or R_(3a) and R_(3a)′ together form a group offormula —N(R_(33a′)), wherein R_(33a′) represents a group of formulaR_(33′) or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m).

In a second preferred embodiment of the process of embodiment (4) thecompound comprising a moiety of formula (II) is a compound of formula(IIa)

wherein:

-   -   either:        -   R_(3a) represents a group of formula R₃ or a group of            formula F₂ or -L(F₂)_(m)(Z)_(n-m) and R_(3a)′ independently            represents a group of formula R₃′ or a group of formula F₂            or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula —O— or            —N(R_(33a′)), wherein R_(33a′) represents a group of formula            R_(33′) or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m); or        -   R_(3a) and R_(3a)′ together form a group of formula            —N(R_(33a′))—N(R_(33a′))—, wherein each R_(33a′) is the same            or different and represents a group of formula R_(33′) or a            group of formula F₂ or -L(F₂)_(m)(Z)_(n-m);    -   m is an integer having a value of from zero to n;    -   R₁ is as hereinbefore defined, and wherein R₁ comprises at least        a first thiol group and a second thiol group, said first thiol        group being attached to the 2-position in the compound of        formula (IIIa) and second thiol group being attached to the        3-position in the compound of formula (IIIa);    -   F₂, X, X′, R₃, R₃′, R_(33′), L, Z and n are all as herein        defined; and    -   step (ii) further involves cleaving the bond between the group        R₁ and the carbon atom at the 3-position of the moiety of        formula (IIIa).

In this second preferred embodiment, R_(3a) preferably represents agroup of formula R₃ or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m) andR_(3a)′ independently represents a group of formula R₃′ or a group offormula F₂ or -L(F₂)_(m)(Z)_(n-m); or R_(3a) and R_(3a)′ together form agroup of formula —N(R_(33a′)), wherein R_(33a′) represents a group offormula R_(33′) or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m).

Accordingly, this second preferred embodiment provides a process forcleaving a cross-linker moiety from a functional moiety which containsat least two thiol groups. An example of such a functional moiety is amoiety which contains a disulfide group, such as a protein containingtwo cysteine residues which are linked to one another via a disulfidebridge.

Preferably, in the process of embodiment (4) the compound comprising amoiety of formula (II) also comprises at least one functional moiety offormula F₂.

In the process of embodiment (4), the step (ii) of cleaving the bond(s)to the group of formula R₁ can be carried out using routine methods forcleaving a thiol bond at an unsaturated carbon centre, for example usingroutine methods for cleaving a thiol attached to an electron deficientalkene.

Preferably, step (ii) of the process of embodiment (4) is effected byincubating the compound with a reagent that is capable of acting as anucleophile in a Michael reaction. Examples of reagents that are wellknown to be capable of acting as a nucleophile in a Michael reactioninclude phosphine compounds, phosphite compounds, thiols, selenols,amines and soft carbon nucleophilic compounds. Phosphine compounds andphosphite compounds both contain a trivalent phosphorous atom. In aphosphine, the phosphorous atom is attached to hydrogen or carbon atoms,while in a phosphite the phosphorous atom is attached to oxygen atoms(it being understood that the carbon atoms and oxygen atoms arethemselves further attached to other groups in the respectivecompounds). Thiols are organic compounds containing a thiol group —SH.Selenols are organic compounds containing an —SeH group. Amines arecompounds containing an amine functional group. Soft carbon nucleophilesare compounds which contain a soft nucleophilic carbon centre. Exemplarysoft carbon nucleophiles are disclosed in U.S. Pat. No. 5,414,074, thecontent of which is herein incorporated by reference in its entirety.Those of skill in the art would of course be able to select appropriatereagents that are capable of acting as a nucleophile in a Michaelreaction as a matter of routine, for example by routinely selecting asuitable reagent from amongst the exemplified list of classes of reagentherein described.

Presently preferred reagents are phosphine compounds and thiols. Aparticularly preferred phosphine is tris(2-carboxyethyl)phosphine, whichis commonly known as “TCEP” and is commonly used in the art to reducedisulfide bonds in compounds, for example, in proteins.Tris(2-carboxyethyl)phosphine can also be supplied in the form of asalt, such as its hydrochloride salt. A particularly preferred thiol isglutathione. Further preferred thiols are 1,2-ethanedithiol,2-mercaptoethanol and dithiothreitol (i.e., HSCH₂CH(OH)CH(OH)CH₂SH,commonly known as DTT). A preferred group of reagents is1,2-ethanedithiol, 2-mercaptoethanol, dithiothreitol, glutathione andtris(2-carboxyethyl)phosphine.

For the avoidance of doubt, as used herein, the term “reagent that iscapable of acting as a nucleophile in a Michael reaction” means areagent that is capable of reacting with an α,β-unsaturated moiety in acompound, and in particular a moiety of formula (V)

wherein X is as herein defined. Such reagents are sometimes known as“reagents that are capable of acting as a nucleophile in a conjugateaddition reaction”. Clearly, the reagents are not limited to reagentswhich react through a nucleophilic carbon centre (e.g., soft carbonnucleophiles), but also include reagents which react through anucleophilic non-carbon centre, such as the exemplary reagents that aredescribed herein.

The present invention also provides a process which comprises:

-   (i) carrying out a process for producing a conjugate as defined in    embodiment (2) or (3); and-   (ii) subsequently regenerating the compound of formula R₁—H from    said conjugate.

Typically, in this process the step (ii) is effected by incubating thecompound with a reagent that is capable of acting as a nucleophile in aMichael reaction, for example a reagent as defined in connection withembodiment (4).

The methodology of the present invention also gives rise to a series ofnew compounds, which constitute embodiments (5), (6), (7), (8) and (9)of the present invention.

Embodiment (5) of the present invention relates to a compound of formula(IIa). This compound comprises (a) the moiety of formula (I) and (b) atleast one moiety of formula F₂ linked thereto. Accordingly, it will beclear that this compound can be used to functionalise a thiol-containingfunctional compound (herein referred to as the compound R₁—H) with amoiety of formula F₂, specifically by using it as a reagent in carryingout the process of embodiment (3).

Preferably in embodiment (5) the compound of formula (IIa) is not anN-phenyl 3,4-dibromomaleimide, wherein the N-phenyl groups issubstituted or unsubstituted.

Preferably in the compound of formula (IIa) of embodiment (5) eitherR_(3a) represents a group of formula R₃ or a group of formula F₂ or-L(F₂)_(m)(Z)_(n-m) and R_(3a)′ independently represents a group offormula R₃′ or a group of formula F₂ or -L(F₂)_(m)(Z)_(n-m) or R_(3a)and R_(3a)′ together form a group of formula —N(R_(33a′)), whereinR_(33a′) represents a group of formula R_(33′) or a group of formula F₂or -L(F₂)_(m)(Z)_(n-m).

Embodiment (6) of the present invention is directed to a compound offormula (IIb), provided that that R₃ and R₃′ do not together form agroup of formula —N(R_(33′)). For the avoidance of doubt, therefore, inthis compound, R₃ and R₃′ are the same or different and each representsa hydrogen atom or a group of formula E, Nu, -L(Z)_(n) or IG or R₃ andR₃′ together form a group of formula —O— or —N(R_(33′))—N(R_(33′))—,wherein each R_(33′) is the same or different and represents a hydrogenatom or a group of formula Y, Nu, -L(Z)_(n) or IG. Preferably R₃ and R₃′are the same or different and each represents a hydrogen atom or a groupof formula E, Nu, -L(Z)_(n) or IG.

The compound of embodiment (6) thus constitutes an intermediate obtainedby carrying out step (i) of the process of embodiment (2) of the presentinvention, and specifically it is an intermediate which carries thefirst functional moiety attached to the reactive cross-linking reagentof the present invention. This intermediate can then readily beconverted into a conjugate molecule further comprising the secondfunctional moiety by carrying out step (ii) of the process of embodiment(2) of the present invention.

Embodiment (7) of the present invention relates to a compound of formula(III), which comprises at least one group of formula F₂ and in whichR_(2a) is not a hydrogen atom. Clearly, therefore, this compoundconstitutes a conjugate obtainable according to the use and processes ofthe present invention, which comprises both the first functional moietyand the second functional moiety cross-linked via the cross-linkingmoiety of the present invention. One experiment described in Hong et al.(J. Am. Chem. Soc., 2009, 131 (29), pp 9986-9994) uses a substituted7-oxanorbornadiene moiety as a cross-linker and generates a conjugatecontaining a maleimide cross-linker. However, this methodologynecessarily generates a hydrogen atom at the substituent positioncorresponding to the R_(2a) substituent. In contrast, it will beimmediately clear to those of skill in the art that the methodology ofthe present invention can readily be applied to obtain an R_(2a)substituent other than hydrogen, simply by selecting a group other thana hydrogen atom to be attached to the moiety of formula (I) whencarrying out a suitable process to synthesise the conjugate. Thecompound of formula (III) according to embodiment (7) can thus be used,for example, to effect further functionalisations (where R_(2a) is anelectrophilic leaving group, a nucleophilic group or a linker groupcarrying a reactive group). Alternatively, the second functional moietyF₂ may itself be located at this substituent position (either beingdirectly attached to the 3-position or being attached thereto via alinker group). Still further, the group of formula R_(2a) may constitutean inert group of formula IG, for example a bulky, chemically unreactivesubstituent which discourages further reactions from occurring to theconjugate molecule.

Preferably in embodiment (7) the compound of formula (III) is not acompound of formula (N):

wherein

R_(N1) and R_(N2) are independently selected from hydrogen, amino,hydroxy, cyano, nitro, carboxylate, carboxamide, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclic aryl, optionally substitutedheteroaryl, optionally substituted alkoxy, optionally substitutedmercaptoalkyl, optionally substituted mono- or di-alkyl amino,optionally substituted cycloalkyl, optionally substitutedheteroalicyclic, or optionally substituted aminoalkyl:

R_(N3) is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclic aryl,optionally substituted heteroaryl, optionally substituted cycloalkyl, oroptionally substituted heteroalicyclic group; and pharmaceuticallyacceptable salts thereof.

Embodiment (8) of the present invention is directed to a compound offormula (IIIa). In particular, this compound comprises a firstfunctional moiety having at least a first and a second thiol group whichare attached to the cross-linker reagent. The compound optionallyfurther comprises at least one additional functional moiety. Preferably,the compound of formula (IIIa) comprises at least one functional moietyof formula F₂. Preferably, the group of formula R₁ is a peptide orprotein comprising at least two cysteine residues, for example twocysteine residues which in the unbound peptide or protein typically forman internal disulfide bridge in the peptide or protein.

Embodiment (9) of the present invention relates to a compound of formula(IVa) or (IVb). It will be appreciated that these compounds constitute aconjugate molecule since they comprise both a first functional moietyand a second functional moiety and furthermore that they comprise asingle, rather than a double, carbon-carbon bond between the 2-positionand the 3-position. However, unlike conjugates prepared usingconventional maleimide reagents, the compounds of formula (IVa) and(IVb) carry a total of at least two functional moieties at the 2- and3-positions.

The compounds of formula (IVa) and (IVb) can be prepared usingstraightforward methods. In one such method, the conjugate molecule isprepared by carrying out a process of embodiment (2) of the presentinvention, in which the step (ii) involves an electrophilic additionreaction of F₂ across the carbon-carbon double bond between the2-position and the 3-position of the formula (II). In another method, aconjugate is firstly prepared which still contains the carbon-carbondouble bond between the 2-position and the 3-position and then anelectrophilic addition reaction is carried out to saturate the doublebond. This electrophilic addition reaction may involve the addition of afurther functional moiety (for example, a thiol-containing furtherfunctional moiety). Alternatively, it may involve any other reagentroutinely used to carry out electrophilic addition reactions atunsaturated carbon-carbon centres. For example, the reagent may be ahydrogen halide, a dihalogen, sulfuric acid, water, an alcohol, H₂S, amercaptan or a carboxylic acid.

Embodiment (10) of the present invention relates to a process forproducing a compound of formula (IVa) or (IVb). It will be appreciatedthat the position at which the group R₄ from the compound of formulaR₄—H adds to the compound of formula (III) will depend on the precisestructure of the compound of formula (III), the nature of the reagentR₄—H and the reaction conditions under which the reaction is carriedout. Usually, the group of formula R₄ will add to the carbon atom whichis capable of forming the most stable cationic intermediate uponaddition of a proton to the compound of formula (III) (i.e., inaccordance with Markovnikov's rule). A person skilled in the art wouldappreciate that if a specific location is desired for addition of thegroup of formula R₄, routine selection of the reaction conditions andthe identity of the other groups on the compound of formula (III) may becapable of achieving such regioselectivity.

As will be clear to those of skill in the art, the methodology of thepresent invention is broadly applicable to known processes and methodswhich involve conjugation of functional moieties. Typically,conventional processes and methods of this type can straightforwardly bemodified by replacing a conventionally known thiol-reactive group on across-linking molecule which links together two functional molecules(such as a maleimide group) by the moiety of formula (I) of the presentinvention.

Examples of routine processes include processes for detecting asubstance, particularly a substance of biological interest such as anantigen or a DNA, processes for purifying a thiol compound containing afunctional moiety and assay processes for identifying whether asubstance interacts with such a compound. Accordingly, the presentinvention also provides the following embodiments (11), (12) and (13),which are directed to detection, purification and assay processescarried out in accordance with the methodology of the present invention.

Embodiment (11) relates to a process for detecting whether a substanceis present in a sample. Typically, the substance is a substance ofbiological interest, for example an antigen, an antibody, a DNA or anRNA. A compound of the present invention is incubated with the sample.This compound is a conjugate which comprises at least two functionalmoieties: firstly, a functional moiety that is capable of generating adetectable signal and secondly a functional moiety that is capable ofinteracting with the substance under test. The functional moiety that iscapable of generating a detectable signal is most preferably an enzyme,but can also be, for example, a detectable moiety or an affinity tag.Clearly, the nature of the functional moiety that is capable ofinteracting with the substance under test depends on the nature of thesubstance itself. For example, where the substance is an antigen, thisfunctional moiety is typically an antibody. Where the substance is a DNAor an RNA, this functional moiety is typically a complementary strand ofDNA or RNA, where “complementary” means that the functional moiety iscapable of interacting with the substance (i.e., hybridising to it).

Preferably, the step of incubating is followed by a step of removing anyamount of conjugate that has not interacted with (i.e., bound to) thesubstance under test, for example a step of washing. This can beachieved, for example, by employing an assay in which the substance isattached to a solid substrate (for example, via an interaction betweenthe substance and a further functional moiety which is (a) capable ofinteracting with the substance and (b) attached to the substrate). Inthat case, non-interacting conjugate can readily be removed by washingwhile retaining conjugate that is bound to the substance under test.

The process of embodiment (11) also comprises a step of monitoring for asignal under conditions allowing for generation of a detectable signalfrom said functional moiety that is capable of generating a detectablesignal. For example, where this functional moiety is an enzyme, thisstep comprises adding a substrate for the enzyme which generates adetectable signal when it is turned over by the enzyme (such asgenerating a coloured or fluorescent product). Preferred enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactrosidase andglucose oxidase.

Preferably the process of embodiment (11) constitutes an ELISA(“enzyme-linked immunosorbent assay”) process, a LAB (“labelledavidin-biotin”) assay process or a BRAB (“bridged avidin-biotin”) assayprocess or an ABC (“avidin-biotin complex”) assay process. All of theseassay processes are routine immunoassay processes and would be familiarto those of skill in the art. Most preferably, the process of embodiment(11) constitutes an ELISA process.

Embodiment (12) relates to a process for purifying a compound of formulaR₁—H from a sample. The process comprises incubating the sample with acompound comprising (a) a moiety of formula (I) and (b) at least oneaffinity tag linked thereto, to effect a process according to embodiment(3) and thereby obtain a conjugate comprising the group R₁ and anaffinity tag. The conjugate is then incubated with a compound comprisingat least one affinity tag partner under conditions allowing forpurification of said conjugate from said sample. A particularly suitableaffinity tag is biotin and a particularly suitable affinity tag partneris avidin or streptavidin.

Embodiment (12) is directed to a process for identifying whether asubstance interacts with a functional moiety of formula R₁. The processinvolves the following steps:

-   -   producing a conjugate comprising (a) said functional moiety of        formula R₁, and (b) a detectable moiety which is capable of        producing a signal which can be modified by said substance, by        carrying out a process of embodiment (2) or (3);    -   incubating the conjugate with the substance;    -   obtaining a signal from the detectable moiety; and    -   comparing the signal with a control signal obtainable when the        conjugate has not been contacted with the substance, thus        determining whether the substance interacts with the conjugate.

Förster resonance energy transfer, or “FRET”, assays are an exemplaryembodiment of the process of embodiment (12). In a FRET assay, thedetectable moiety attached to the functional moiety of formula R₁ is adonor chromophore and the substance is labelled with an acceptorchromophore. Donor chromophore/acceptor chromophore pairs are well knownin the art. One example is the cyan fluorescent protein (CFP)/yellowfluorescent protein (YFP) pair. FRET assays can be used, for example, tostudy protein-protein interactions, protein-DNA interactions and proteinconformational changes.

Conjugates of the present invention, specifically those of embodiments(7), (8) and (9) are also suitable for use in methods of medicaltreatment or diagnosis. The present invention therefore provides, in anembodiment (14), use of such a compound in a method of treatment of thehuman or animal body by surgery or therapy or a diagnostic methodpractised on the human or animal body. Embodiment (14) also relates to amethod of treatment of the human or animal body or a diagnostic methodpractised on the human or animal body which comprises administering tothe human or animal body such a compound.

As those skilled in the art would immediately recognise, conjugatessuitable for use in the embodiment (14) are typically those whichcomprise at least one biologically active moiety. In one preferredembodiment, the first functional moiety is a biologically active moietyand the second functional moiety is a polymeric moiety (for example, amoiety capable of enhancing bioavailability and/or stability in vivo,such as a polyethylene glycol moiety) or an antibody (for example, inorder to form an immunotoxin conjugate for use in targeting specificantigens, such as in treatment of cancers). In another preferredembodiment, the conjugate comprises a radioactive moiety and abiologically active moiety, for use in a PET (positron emissiontomography) diagnostic method.

It will be understood that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination and the severity of the particular diseaseundergoing treatment. Optimum dose levels and frequency of dosing willbe determined by clinical trial, but an exemplary dosage would be0.1-1000 mg per day. The medical compounds with which the invention isconcerned may be prepared for administration by any route consistentwith their pharmacokinetic properties. The orally administrablecompositions may be in the form of tablets, capsules, powders, granules,lozenges, liquid or gel preparations, such as oral, topical, or sterileparenteral solutions or suspensions. Tablets and capsules for oraladministration may be in unit dose presentation form, and may containconventional excipients such as binding agents, for example syrup,acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone;fillers, for example lactose, sugar, maize-starch, calcium phosphate,sorbitol or glycine; tabletting lubricant, for example magnesiumstearate, talc, polyethylene glycol or silica; disintegrants, forexample potato starch, or acceptable wetting agents such as sodiumlauryl sulphate. The tablets may be coated according to methods wellknown in normal pharmaceutical practice. Oral liquid preparations may bein the form of, for example, aqueous or oily suspensions, solutions,emulsions, syrups or elixirs, or may be presented as a dry product forreconstitution with water or other suitable vehicle before use. Suchliquid preparations may contain conventional additives such assuspending agents, for example sorbitol, syrup, methyl cellulose,glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, forexample lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles(which may include edible oils), for example almond oil, fractionatedcoconut oil, oily esters such as glycerine, propylene glycol, or ethylalcohol; preservatives, for example methyl or propyl p-hydroxybenzoateor sorbic acid, and if desired conventional flavouring or colouringagents.

For topical application to the skin, the medical compounds may be madeup into a cream, lotion or ointment. Cream or ointment formulationswhich may be used for the drug are conventional formulations well knownin the art, for example as described in standard textbooks ofpharmaceutics such as the British Pharmacopoeia.

For topical application by inhalation, the medical compounds may beformulated for aerosol delivery for example, by pressure-driven jetatomizers or ultrasonic atomizers, or preferably by propellant-drivenmetered aerosols or propellant-free administration of micronizedpowders, for example, inhalation capsules or other “dry powder” deliverysystems. Excipients, such as, for example, propellants (e.g. Frigen inthe case of metered aerosols), surface-active substances, emulsifiers,stabilizers, preservatives, flavourings, and fillers (e.g. lactose inthe case of powder inhalers) may be present in such inhaledformulations. For the purposes of inhalation, a large number of apparataare available with which aerosols of optimum particle size can begenerated and administered, using an inhalation technique which isappropriate for the patient. In addition to the use of adaptors(spacers, expanders) and pear-shaped containers (e.g. Nebulator®,Volumatic®), and automatic devices emitting a puffer spray (Autohaler®),for metered aerosols, in particular in the case of powder inhalers, anumber of technical solutions are available (e.g. Diskhaler®, Rotadisk®,Turbohaler® or the inhalers for example as described in European PatentApplication EP 0 505 321).

For topical application to the eye, the medical compounds may be made upinto a solution or suspension in a suitable sterile aqueous or nonaqueous vehicle. Additives, for instance buffers such as sodiummetabisulphite or disodium edeate, preservatives including bactericidaland fungicidal agents such as phenyl mercuric acetate or nitrate,benzalkonium chloride or chlorhexidine, and thickening agents such ashypromellose may also be included.

The active ingredient may also be administered parenterally in a sterilemedium. Depending on the vehicle and concentration used, the drug caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as a local anaesthetic, preservative and buffering agentscan be dissolved in the vehicle.

The medical compounds of the invention may be used in conjunction with anumber of known pharmaceutically active substances.

The present invention further provides a compound containing a moiety offormula (VI) and a functional moiety linked thereto

wherein:

-   -   X and X′ are the same or different and each represents oxygen,        sulfur or a group of formula ═NQ, in which Q is hydrogen,        hydroxyl, C₁₋₆ alkyl or phenyl; and    -   said functional moiety is selected from a detectable moiety, an        enzymatically active moiety, an affinity tag, a hapten, an        immunogenic carrier, an antibody or antibody fragment, an        antigen, a ligand, a biologically active moiety, a liposome, a        polymeric moiety, an amino acid, a peptide, a protein, a cell, a        carbohydrate, a DNA and an RNA.

Typically said functional moiety is linked to the 5-position or6-position of the moiety of formula (VI). Thus, the compound can beproduced by reacting a functional moiety containing an alkene moietywith the carbon-carbon double bond between the 2- and 3-positions of acompound containing a moiety of formula (I) or (II), or a compound offormula (Ia), (Ib), (IIa), (IIb), (III) or (IIIa), in a photocatalytic[2+2] cycloaddition reaction.

Preferably the moiety of formula (VI) comprises at least one, forexample one, further functional moiety which is itself independentlyselected from a detectable moiety, an enzymatically active moiety, anaffinity tag, a hapten, an immunogenic carrier, an antibody or antibodyfragment, an antigen, a ligand, a biologically active moiety, aliposome, a polymeric moiety, an amino acid, a peptide, a protein, acell, a carbohydrate, a DNA and an RNA. In this embodiment, preferablyone such further functional moiety carries a thiol moiety and is linkedto the 2-position of the moiety of formula (VI) via the sulfur atom ofsaid thiol moiety. Thus, this compound can be produced from a compoundcontaining a moiety of formula (I) by:

-   -   reacting the compound containing a moiety of formula (I) with        said further functional moiety carrying a thiol moiety, thus        creating an intermediate product which is a compound containing        a moiety of formula (II); and    -   reacting this intermediate compound with a functional moiety        containing an alkene moiety to effect a photocatalytic        [2+2]cycloaddition reaction between said alkene moiety and the        carbon-carbon double bond between the 2- and 3-positions of the        intermediate product.

Preferably, the compound containing a moiety of formula (VI) and afunctional moiety linked thereto is a compound of formula (VIa)

wherein:

-   -   X and X′ are the same or different and each represents oxygen,        sulfur or a group of formula ═NQ, in which Q is hydrogen,        hydroxyl, C₁₋₆ alkyl or phenyl;    -   YR₁ is a group of formula Y or R₁:    -   Y is an electrophilic leaving group;    -   R₁ is a group of formula —F₁ or —S-L-F₁, wherein R₁ carries a        thiol moiety and is linked to the 2-position of the moiety of        formula (VIa) via the sulfur atom of said thiol moiety;    -   R_(2a), R_(3a) and R_(3a)′ are each as defined in relation to        the compound of formula (IIa);    -   Each of R_(alk1), R_(alk2), R_(alk3) and R_(alk4) is the same or        different and is a group of formula R_(2a), with the proviso        that at least one of R_(alk1), R_(alk2), R_(alk3) and R_(alk4)        contains a group of formula F₂; and    -   F₁ and any group of formula F₂ are the same or different and are        each selected from a detectable moiety, an enzymatically active        moiety, an affinity tag, a hapten, an immunogenic carrier, an        antibody or antibody fragment, an antigen, a ligand, a        biologically active moiety, a liposome, a polymeric moiety, an        amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA        and an RNA.

Preferably YR₁ is a group of formula R₁, with R₁, preferably being agroup of formula —F₁. Preferably the compound of formula (VIa) comprisesone group of formula F₂.

EXAMPLES

The following Examples illustrate the scientific principles underlyingthe present invention. Many of the Examples are Reference Examples sincethey do not involve linkage of two functional moieties. However, linkageof a functional moiety to linking groups relevant to the invention,cleavage of the functional moieties therefrom, and linkage of afunctional moiety, via linking groups relevant to the invention, tonumerous other secondary moieties (including other functional moieties)has been exhaustively demonstrated. A large degree of structuralvariation is shown to be readily tolerated, evidencing the broadapplicability of the present invention.

A) Preliminary Examples

¹H and ¹³C NMR spectra were recorded at room temperature on a BrukerAvance 500 instrument operating at a frequency of 500 MHz for ¹H and 125MHz for ¹³C. ¹H NMR spectra were referenced to the CDCl₃ (7.26 ppm)signal. ¹³C NMR spectra were referenced to the CDCl₃ (77.67 ppm) signal.

Infra-red spectra were run on a PerkinElmer Spectrum 100 FT-IRspectrometer operating in ATR mode with frequencies given in reciprocalcentimeters (cm⁻¹). Mass spectra and high resolution mass data wererecorded on a VG70-SE mass spectrometer (EI mode and CI mode).

Melting points (m.p.) were taken on a Gallenkamp heating block and areuncorrected. Optical rotation measurements were carried out using aPerkinElmer 343 polarimeter with a cell length of 10 cm.

Abbreviations

-   Boc Tert-butyloxycarbonyl group.-   Cys Cysteine-   Mal Maleimide-   DMF Dimethylformamide-   TCEP (tris(2-carboxyethyl)phosphine)-   LC-ESI-MS Liquid chromatography electron spray ionisation mass    spectroscopy-   Pn GrB2-SH2 domain L111C (used as a model protein)-   Gc Glucose

Reference Example 1: Preparation of Bromomaleimide (Compound 1)

To maleimide (2.00 g, 0.02 mol) in chloroform (15 mL) was added bromine(1.16 mL, 0.02 mol) in chloroform (15 mL) dropwise. The reaction mixturewas refluxed for 2 hours and left to cool to room temperature over 1hour. Solid yellow precipitate was filtered off and washed with coldchloroform (2×50 mL) to afford white crystals of crude2,3-dibromosuccinimide (4.09 g, 0.016 mol). The crude succinimide wasdissolved in tetrahydrofuran (50 mL) and triethylamine (2.4 mL, 0.017mol) in tetrahydrofuran (10 mL) was added over 5 minutes at 0° C. Thereaction mixture was allowed to warm to room temperature and stirred for48 hours. The solid was filtered off and washed with tetrahydrofuran (50mL) to afford a pale yellow powder (2.14 g, 0.012 mol) in 59% yield.

¹H NMR (500 MHz, CDCl₃): δ=7.67 (br s, 1H, NH), 6.89 (s, 1H, C═CH); ¹³CNMR (125 MHz, CDCl₃): δ=173.8 (C═O), 170.5 (C═O), 136.9 (—(Br)C═C—),135.4 (—C═CCH—); IR (solid, cm⁻¹): 3235 (s), 1709 (s); MS (CI+) m/z,(%): 178 (⁸¹M+, 32), 176 (⁷⁹M+, 32), 125 (25), 86 (100); Mass calculatedfor C₄H₃O₂N⁷⁹Br: 175.93472. Found: 175.93493; m.p. 148-151° C.

Reference Example 2: Preparation of N-methylbromomaleimide (Compound 2)

To N-methyl maleimide (0.5 g, 4.5 mmol) in methanol (22.5 mL) was addedbromine (0.52 mL, 10 mmol) dropwise. The reaction mixture was stirred atroom temperature for 24 hours. Solvent was removed in vacuo and thereaction mass was dissolved in tetrahydrofuran (20 mL) and triethylamine(0.8 mL, 5.85 mmol) added, then stirred for 24 hours at roomtemperature. The material was purified by flash chromatography on silicagel (petroleum ether:ethyl acetate, 7:3) to afford a pale white powder(0.761 g, 4.0 mmol) in 89% yield.

¹H NMR (500 MHz, CDCl₃): δ=6.90 (s, 1H, C═CH), 3.09 (s, 3H, N—CH₃); ¹³CNMR (125 MHz, CDCl₃): δ=168.6 (C═O), 165.4 (C═O), 131.9 (—C═CH—), 131.4((Br)C═C—), 24.7 (—N—CH₃); IR (solid, cm⁻¹): 3106 (s), 1708 (s); MS (CI)m/z, (%): 192 (⁸¹M+, 99), 190 (⁷⁹M+, 100); Mass calculated forC₅H₅O₂N⁷⁹Br 189.95037. Found: 189.95052; m.p: 77-79° C.

Reference Example 3: Preparation of N-Boc-Cys(Mal)-OMe (Compound 4)

To a stirring solution of N-Boc-Cys-OMe (compound 3) (36 mg, 0.153 mmol)and sodium acetate (13 mg, 0.153 mmol) in methanol (3 mL) was addedbromomaleimide (30 mg, 0.169 mmol) in methanol (3 mL). After 1 minutesolvent was removed in vacuo. The material was purified by flashchromatography on silica gel (petroleum ether ethyl acetate, gradientelution from 9:1 to 7:3) to afford a pale yellow powder (51 mg, 0.153mmol) in 100% yield.

¹H NMR (500 MHz, CDCl₃): δ=7.63 (s, 1H, NH), 6.27 (s, 1H, C═CH), 5.40(d, 1H, J=6.8, NH), 4.67 (ddd, 1H, J=6.8, 5.4 and 5.1, —HN—CH—C(O)—),3.80 (s, 3H, O—CH₃), 3.48 (dd, 1H, J=13.8 and 5.1, —S—CHH—), 3.62 (dd,1H, J=14.1 and 5.4, —S—CHH—) 1.45 (s, 9H, 3×CH₃); ¹³C NMR (125 MHz,CDCl₃): δ=170.2 (C═O), 168.9 (C═O), 167.6 (C═O), 155.2 (C═O), 155.9(—C═CH—), 119.7 (—C═CH—), 81.1 ((CH₃)CO—), 53.3 (O—CH₃), 52.7 (CH), 34.0(CH₂), 28.3 (3×CH₃); IR (solid, cm⁻¹) 3236 (w), 1715 (s); MS (CI+) m/z,(%): 331 (M+H, 5), 275 (20), 231 (100); Mass calculated for[C₃H₁₈O₆N₂S]+H; 331.09638. Found: 331.09684; ²⁰α_(D): −41.9° (c=1.0,Methanol); m.p. 145-147° C.

Reference Example 4: Preparation of N-Boc-Cys(N-Me-Mal)-OMe (Compound 5)

To a stirring solution of N-Boc-Cys-OMe (32 mg, 0.136 mmol) in methanol(4 mL) was added sodium acetate (82 mg, 0.408 mmol). To this was addedN-methyl bromomaleimide (25.8 mg, 0.136 mmol) in methanol (4 mL) over 10minutes. The reaction turned light yellow. The solvent was removed invacuo and the residue was purified by flash chromatography on silica gel(petroleum ether ethyl acetate, gradient elution from 9:1 to 7:3) toafford a pale white powder (39.3 mg, 0.114 mmol) in 84% yield.

¹H NMR (500 MHz, CDCl₃): δ=6.26 (s, 1H, C═CH), 5.36 (d, 1H, J=6.3, NH),4.66 (m, 1H, —HN—CH—), 3.79 (s, 3H, O—CH₃), 3.46 (dd, 1H, J=5.2 and 5.0,—S—CHH—), 3.35 (dd, 1H, J=13.7 and 5.1, —S—CHH—), 3.00 (s, 3H, —N—CH₃),1.44 (s, 9H, 3×CH₃); ¹³C NMR (125 MHz, CDCl₃): δ=170.2 (C═O), 169.5(C═O), 167.9 (C═O), 155.0 (C═O), 149.9 (—C═CH—), 118.7 (—C═CH—), 80.9((CH₃)₃CO—), 53.1 (O—CH₃), 52.7 (CH), 33.8 (CH₂), 28.3 (3×CH₃), 24.1(—N—CH₃); IR (solid, cm⁻¹) 3367.8, 2977.1, 1694.7; MS (ES+) m/z, (%):367(46), 311 (M, 100); Mass calculated for C₁₄H₂₀N₂O₆NaS: 367.0940.Found: 367.0931; ²⁰α_(D): −18.55° (C=1.0, Methanol); m.p. 101-103° C.

Reference Example 5: Preparation of N-Boc-Cys(Succ)-OMe (Compound 6)

To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.153 mmol) in methanol(3 mL) was added maleimide (17 mg, 0.169 mmol) in methanol (3 mL). After1 minute solvent was removed in vacuo. The material was purified byflash chromatography on silica gel (dichloromethane:methanol, gradientelution from 99:1 to 7:3) to afford a colourless oil (51 mg, 0.153 mmol)in 100% yield that was a 1:1 mixture of diastereomers.

¹H NMR (500 MHz, CDCl₃): δ=9.00 (s, 1H, NH), 8.95 (s, 1H, NH), 5.59 (1H,d, J=7.6, NH), 5.41 (d, 1H, J=7.6, NH), 4.65-4.56 (m, 2H,2×—HN—HC—C(O)—) C═CHH), 3.93 (dd, 1H, J=9.3 and 3.9, CH), 3.86 (dd, 1H,J=9.2 and 4.2, CH), 3.76 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.51 (dd,1H, J=13.8 and 4.6, —CHH—), 3.36 (dd, 1H, J=14.1 and 6.0, —CHH—),3.19-3.11 (m, 3H, 3×—CHH—), 2.96 (dd, 1H, J=13.1 and 7.1, —CHH—),2.54-2.02 (m, 2H, —CHH—) 1.43 (s, 18H, 9×CH₃); ¹³C NMR (125 MHz, CDCl₃):δ=177.2 (C═O), 177.1 (C═O), 175.1 (C═O), 175.0 (C═O), 172.0 (C═O), 171.5(C═O), 155.5 (C═O), 155.3 (C═O), 80.6 (2×—OCCH₃), 53.6 (CH), 52.91(OCH₃), 52.85 (OCH₃), 50.8 (CH), 40.6 (CH), 40.0 (CH), 37.3 (CH₂), 37.0(CH₂), 34.6 (CH₂), 34.1 (CH₂), 28.3 (6×CH₃); IR (oil, cm⁻¹) 3233 (w),2980 (w), 1783 (w), 1709 (s); MS (CI+) m/z, (%): 333 (M+H, 15), 277(50), 233 (100); Mass calculated for C₁₃H₂₀O₆N₂S: 332.10420. Found:332.10475.

Reference Example 6: Demonstration that Maleimide does not DisplaceThiol from N-Boc-Cys(Mal)-OMe and that Bromomaleimide does not DisplaceThiol from N-Boc-Cys(Succ)-OMe

To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.153 mmol) and sodiumacetate (13 mg, 0.153 mmol) in methanol (3 mL) was added bromomaleimide(30 mg, 0.169 mmol) in methanol (3 mL). After 10 minutes maleimide (17mg, 0.169 mmol) was added. Solvent was removed in vacuo and ¹H NMRanalysis showed only compound 4 and unreacted maleimide.

To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.153 mmol) and sodiumacetate (13 mg, 0.153 mmol) in methanol (3 mL) was added maleimide (17mg, 0.169 mmol) in methanol (3 mL). After 10 minutes bromomaleimide (30mg, 0.169 mmol) was added. Solvent was removed in vacuo and ¹H NMRanalysis showed only compound 6 and unreacted bromomaleimide.

Reference Example 7: Competition Reaction Between Bromomaleimide andMaleimide for N-Boc-Cys-OMe

To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.153 mmol) and sodiumacetate (13 mg, 0.153 mmol) in methanol (3 mL) was added a mixture ofbromomaleimide (30 mg, 0.169 mmol) and maleimide (17 mg, 0.169 mg) inmethanol (3 mL). After 1 minute solvent was removed in vacuo. Thematerial was purified by flash chromatography on silica gel (petroleumether:ethyl acetate, gradient elution from 9:1 to 7:3) to afford a paleyellow powder 4 (36 mg, 0.108 mmol) in 70% yield and a colourless oil 6(15 mg, 0.045 mmol) in 30% yield.

Reference Example 6 demonstrated that, once attached to a succinimide ormaleimide moiety, the cysteine moiety is not capable of detaching in thepresence of these reagents. Reference Example 7 therefore demonstratesthat the cysteine reagent reacts more rapidly with bromomaleimide thanwith maleimide (i.e., the reaction kinetics are more favourable forformation of compound 4).

Reference Example 8: Demonstration of Selectivity of the BromomaleimideReagent for N-Boc-Cys-OMe Compared to Propylamine

To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.153 mmol) andpropylamine (10 μL, 0.153 mmol) in methanol (3 mL) was addedbromomaleimide (30 mg, 0.169 mmol) in methanol (3 mL). After 1 minutesolvent was removed in vacuo. The material was purified by flashchromatography on silica gel (petroleum ether:ethyl acetate, gradientelution from 9:1 to 7:3) to afford a pale yellow powder (51 mg, 0.153mmol) in 100%. Data matched those obtained above for N-Boc-Cys(Mal)-OMe4.

Example 1: Cleavage of N-Boc-Cys(Mal)-OMe to Regenerate N-Boc-Cys-OMe

To a stirring solution of 4 (50 mg, 0.151 mmol) in dimethylformamide (2mL) was added 20 ml, of an aqueous buffer (150 mM NaCl, 100 mM NaH₂PO₄,pH 8.0). Tris(2-carboxyethyl)phosphine (430 mg 1.51 mmol) in 20 mL of anaqueous buffer (150 mM NaCl, 100 mM NaH₂PO₄, pH 8.0) was added to thesolution. After 5 minutes the aqueous solution was extracted with ethylacetate (3×25 mL), washed with saturated lithium chloride solution (5×25mL), water (25 mL) and brine (25 mL) and dried over MgSO₄. Solvent wasremoved in vacuo to afford a colourless oil (34.5 mg, 0.148 mmol) in 98%yield. ¹H and ¹³C NMR of this oil showed it to be the commerciallyavailable N-Boc-cysteine methyl ester 3.

Example 2: Reaction of 2,3-Dibromomaleimide with Somatostatin

Somatostatin is peptide hormone which is known to exist in a form inwhich two cysteine residues within the molecule are attached via adisulfide bridge.

1 mg of lyophilised somatostatin (Sigma-Aldrich) was resolubilised in 2ml of 50 mM NaHPO₄ ⁻, pH 6.2, 40% MeCN, 2.5% DMF. 500 μl weretransferred to a Eppendorf reaction tube and diluted in the same bufferto a final concentration of 0.25 mg/ml (152.6 μM), 2.0 equivalents ofTCEP (100× stock solution in 50 mM NaHPO₄ ⁻, pH 6.2, 40% MeCN) wereadded and the reaction incubated for 1 hour at ambient temperature.After reduction of the disulfide bond 1.4 equivalents of2,3-dibromomaleimide (Sigma-Aldrich, 100× stock solution in 50 mMNaHPO₄, pH 6.2, 40% MeCN, 2.5% DMF) were added, the solution gentlymixed and incubated for a further 12 h at 4° C.

Maleimide-bridged somatostatin was detected by LC-ESI-MS (ES⁺/ES⁻).Controls included untreated peptide and somatostatin treated with2,3-dibromomaleimide or TCEP only. Complete reduction was detected bythe reaction of TCEP-treated peptide with maleimide (Sigma-Aldrich, 100×stock solution in 50 mM NaHPO₄ ⁻, pH 6.2, 40% MeCN, 2.5% DMF).

Experimental Data

Untreated somatostatin: [ES+] 1638.04 (m/z 1), 819.82 (m/z 2), 546.95(m/z 3). Maleimide-bridged somatostatin: [ES+]1734.14 Da (m/z 1), 867.40Da (m/z 2), 578.73 (m/z 3).

Reference Example 9: Expression of GrB2-SH2 Domain L111C

The protein GrB2-SH2 domain L111C was used as a model protein. Thismodel protein contains a single cysteine residue.

LC-MS was performed on a Waters Acquity uPLC connected to Waters AcquitySingle Quad Detector (SQD). Column: Acquity uPLC BEH C18 1.7 μm 2.1×50mm. Wavelength: 254 nm. Mobile Phase: 95:5 Water (0.1% Formic Acid):MeCN(0.1% Formic Acid) Gradient over 4 min (to 5:95 Water (0.1% FormicAcid):MeCN (0.1% Formic Acid). Flow Rate: 0.6 ml/min. MS Mode: ES+. ScanRange: m/z=85-2000. Scan time: 0.25 sec. Data obtained in continuummode. The electrospray source of the MS was operated with a capillaryvoltage of 3.5 kV and a cone voltage of 50 V. Nitrogen was used as thenebulizer and desolvation gas at a total flow of 600 L/h. Total massspectra were reconstructed from the ion series using the MaxEnt 1algorithm preinstalled on MassLynx software.

The model protein was over-expressed in E. coli, and the hexa-His-taggedprotein purified using both Ni-affinity chromatography andsize-exclusion chromatography via standard techniques. Analysis usingLC-MS showed a single protein species of mass 14169 which corresponds tothe desired protein.

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added Ellman'sreagent (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexedfor 1 s and maintained at 0° C. for 10 mins, after which the mixture wasanalysed by LC-MS. Analysis showed that a single reaction had occurredyielding a single product with a mass of 14366 showing that C111 wasavailable for functionalisation.

Reference Example 10: Reaction of GrB2-SH2 Domain L111C withBromomaleimide

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide(5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 0° C. for 1 h. Analysis using LC-MS showed a singleprotein species of mass 14265 which corresponds to the desired protein.

The mixture was treated with Ellman's reagent (5 μL, 282 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 10 mins after which the mixture was analysed by LC-MS. Analysisshowed that no reaction with Ellman's reagent was evident, highlightingthat bromomaleimide functionalisation had occurred at C111.

Reference Example 11: Reaction of GrB2-SH2 Domain L111C withN-Methylbromomaleimide

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedN-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed a single protein species of mass 14278 which corresponds to thedesired protein.

The mixture was treated with Ellman's reagent (5 μL, 282 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 00° C.for 10 mins after which the mixture was analysed by LC-MS. Analysisshowed that no reaction with Ellman's reagent was evident, highlightingthat N-methylbromomaleimide functionalisation had occurred at C111.

Example 3: Phosphine-Mediated Reductive Cleavage of GrB2-SH2 DomainL111C/Bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide(5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 0° C. for 1 h. Analysis using LC-MS showed a singleprotein species of mass 14265 which corresponds toprotein/bromomaleimide adduct.

The mixture was treated with TCEP.HCl (5 μL, 282 mM solution in H₂O) at0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 hafter which the mixture was analysed by LC-MS. Analysis showed that theprotein/bromomaleimide adduct had been cleanly cleaved yielding GrB2-SH2domain L111C (mass=14168) in 85% yield. The remaining material wasunreacted protein/bromomaleimide adduct.

Example 4: Phosphine-Mediated Reductive Cleavage of GrB2-SH2 DomainL111C/N-methylbromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedN-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed a single protein species of mass 14278 which corresponds toprotein/N-methylbromomaleimide adduct.

The mixture was treated with TCEP.HCl (5 μL, 282 mM solution in H₂O) at0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 hafter which the mixture was analysed by LC-MS. Analysis showed that theprotein/N-methylbromomaleimide adduct had been cleanly cleaved yieldingGrB2-SH2 domain L111C (mass=14168) in 85% yield. The remaining materialwas unreacted protein/N-methylbromomaleimide adduct.

Example 5: Synthesis of GrB2-SH2 DomainL111C/bromomaleimide/2-Mercaptoethanol Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide(5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 0° C. for 1 h. Analysis using LC-MS showed a singleprotein species of mass 14265 which corresponds toprotein/bromomaleimide adduct.

The mixture was treated with 2-mercaptoethanol (5 μL, 2.82 mM solutionin H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0°C. for 3 h after which the mixture was analysed by LC-MS. Analysisshowed that the protein/bromomaleimide/2-mercaptoethanol adduct had beenformed (mass=14339) in 55% yield. The remaining material was GrB2-SH2domain L111C.

Example 6: Synthesis of GrB2-SH2 Domain111C/N-Methylbromomaleimide/2-Mercaptoethanol Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedN-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed a single protein species of mass 14278 which corresponds toprotein/N-methylbromomaleimide adduct.

The mixture was treated with 2-mercaptoethanol (5 μL, 2.82 mM solutionin H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0°C. for 3 h after which the mixture was analysed by LC-MS. Analysisshowed that the protein/N-methylbromomaleimide/2-mercaptoethanol adducthad been formed (mass=14356) in 61% yield. The remaining material wasGrB2-SH2 domain L111C.

Reference Example 12: Synthesis of GrB2-SH2 DomainL111C/Dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponds toprotein/dibromomaleimide adduct.

Example 7: Synthesis of GrB2-SH2 DomainL111C/Dibromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponds toprotein/dibromomaleimide adduct.

The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2h after which the mixture was analysed by LC-MS. Analysis showed thatthe protein/dibromomaleimide/glutathione adduct was the only proteinspecies present (mass=14572).

Example 8: Glutathione-Mediated Cleavage of GrB2-SH2 DomainL111C/Dibromomaleimide/Glutathione Adduct at Physiological RelevantGlutathione Concentration (5 mM)

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponds toprotein/dibromomaleimide adduct.

The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2h after which the mixture was analysed by LC-MS. Analysis showed thatthe protein/dibromomaleimide/glutathione adduct was the only proteinspecies present (mass=14572).

The mixture was treated with glutathione (5 μL, 100 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4h after which the mixture was analysed by LC-MS. Analysis showed thatGrB2-SH2 domain L111C was the only protein species present (mass=14173).

B) Further Examples

General Procedures

¹H and ¹³C NMR spectra were recorded at room temperature on a BrukerAvance 500 instrument operating at a frequency of 500 MHz for ¹H and 125MHz for ¹³C. ¹H NMR spectra were referenced to the CDCl₃ (7.26 ppm)signal. ¹³C NMR spectra were referenced to the CDCl₃ (77.67 ppm) signal.Infra-red spectra were run on a PerkinElmer Spectrum 100 FT-IRspectrometer operating in ATR mode with frequencies given in reciprocalcentimeters (cm⁻¹). Mass spectra and high resolution mass data for smallmolecules were recorded on a VG70-SE mass spectrometer (EI mode and CImode). Melting points were taken on a Gallenkamp heating block and areuncorrected, 3,4-Dibromomaleimide, lyophilized somatostatin, PEG5000,TCEP and benzeneselenol were purchased from Sigma-Aldrich and usedwithout further purification.

Protein and Peptide Mass Spectrometry

LC-MS was performed on protein samples using a Waters Acquity uPLCconnected to Waters Acquity Single Quad Detector (SQD). Column: AcquityuPLC BEH C18 1.7 μm 2.1×50 mm. Wavelength: 254 nm. Mobile Phase: 95:5Water (0.1% Formic Acid):MeCN (0.1% Formic Acid) Gradient over 4 min (to5:95 Water (0.1% Formic Acid):MeCN (0.1% Formic Acid). Flow Rate: 0.6mL/min. MS Mode: ES+. Scan Range: m/z=85-2000. Scan time: 0.25 sec. Dataobtained in continuum mode. The electrospray source of the MS wasoperated with a capillary voltage of 3.5 kV and a cone voltage of 50 V.Nitrogen was used as the nebulizer and desolvation gas at a total flowof 600 L/h. Total mass spectra were reconstructed from the ion seriesusing the MaxEnt 1 algorithm pre-installed on MassLynx software.MALDI-TOF analysis was performed on a MALDI micro MX (Micromass). Datawas obtained in reflectron positive ion mode with a source voltage of 12kV and a reflectron voltage of 5 kV at a laser wavelength of 337 nm.Samples were prepared as outlined below and those containing peptidewere dialysed for 24 h in deionised H₂O. The peptide and its derivates(0.1-0.3 mg/ml) were spotted onto a MALDI plate in 2 μl sinapinic acid(10 mg/ml) after pre-spotting of trifluoroacetic acid (10 mg/ml). ACTH(10 ng/ml) was used for mass calibration.

Reference Example 13: Preparation of Bromomaleimide

To maleimide (2.00 g, 0.02 mol) in chloroform (15 mL) was added bromine(1.16 mL, 0.02 mol) dropwise in chloroform (15 mL). The reaction mixturewas refluxed for 2 hours and left to cool to room temperature over 1hour. Solid yellow precipitate was filtered off and washed with coldchloroform (2×50 mL) to afford off white crystals of crude2,3-dibromosuccinimide (4.09 g, 0.016 mol). The crude succinimide wasdissolved in tetrahydrofuran (50 mL) and triethylamine (2.4 mL, 0.017mol) in tetrahydrofuran (10 mL) was added over 5 minutes at 0° C. Thereaction mixture was allowed to warm to room temperature and stirred for48 hours. The solid was filtered off and washed with tetrahydrofuran (50mL). Purification by flash chromatography (5% ethyl acetate in petroleumether) afforded the desired compound as a pale yellow powder (2.14 g,0.012 mol) in 59% yield. S (500 MHz, CDCl₃) 7.67 (br s, 1H, NH), 6.89(s, 1H, H-3); δ_(C) (125 MHz, CDCl₃) 173.8 (C═O), 170.5 (C═O), 136.9(C2), 135.4 (C3); IR (solid, cm⁻¹) 3235 (s), 1709 (s); MS (CI+) m/z,(relative intensity): 178 ([⁸¹M+H], 32), 176 ([⁷⁹M+H], 32), 125 (25), 86(100); Mass calcd for [(C₄H₂O₂N⁷⁹Br]+H: 175.9347. Found 175.9349 (CI+);m.p. 148-151° C.; UV (Acetonitrile) ε₂₄₂=13800 and ε₂₇₆=1700 cm⁻¹M⁻¹d³.

Reference Example 14: Preparation of N-Methylbromomaleimide

To N-methylmaleimide (0.5 g, 4.5 mmol) in methanol (10 mL) was addedbromine (232 μL, 4.5 mmol) dropwise in methanol (5 mL). The reactionmixture was stirred at room temperature for 12 hours. The solvent wasremoved in vacuo and dissolved in tetrahydrofuran (20 mL). Triethylamine(815 μL, 5.9 mmol) in tetrahydrofuran (5 mL) was added over 5 minutes,whereupon a precipitate formed. The reaction mixture was stirred for 24hours. The solid was filtered off and washed with tetrahydrofuran (50mL). Purification by flash chromatography (10% ethyl acetate inpetroleum ether) afforded the desired compound as a pale yellow powder(563 mg, 2.96 mmol) in 66% yield. δ_(H) (500 MHz, CDCl₃) 6.90 (s, 1H,H-3), 3.09 (s, 3H, H₃-6); δ_(C) (125 MHz, CDCl₃) 168.6 (C═O), 165.4(C═O), 131.9 (C3), 131.4 (C2), 24.7 (C6); IR (solid, cm⁻¹) 3106 (s),1708 (s); MS (CI+) m/z, (relative intensity): 192 ([⁸¹M+H], 99), 190([⁷⁹M+H], 100); Exact mass calcd for [C₅H₄O₂N⁷⁹Br]+H requires 189.9504.Found 189.9505 (CI+); m.p: 77-79C; UV (Acetonitrile) ε₂₀₉=17100,ε₂₃₈=13200, ε₂₉₉=290 cm⁻¹M⁻¹d³.

Reference Example 15: Preparation of N-Phenylbromomaleimide

To N-phenylmaleimide (2 g, 11.50 mmol) in chloroform (15 mL) was addedbromine (0.65 mL, 12.70 mmol) dropwise in chloroform (5 mL). Thereaction mixture was refluxed for 1 hour, and then allowed to cool toroom temperature. The precipitate was filtered off and washed withchloroform (50 mL). This solid (2.70 g, 8.10 mmol) was dissolved intetrahydrofuran (50 mL) and to this was added dropwise a solution oftriethylamine (1.2 mL, 8.9 mmol) in tetrahydrofuran (10 mL) at 0° C. andthe mixture was stirred for 2 hours. The mixture was allowed to warm toroom temperature and solvent removed in vacuo. The residue was dissolvedin ethyl acetate and washed with H₂O (50 mL) brine (50 mL) and dried(Na₂SO₄). Solvent was removed in vacuo to afford the desired compound asa pale yellow solid (1.80 g, 7.14 mmol) in 62% yield. Data matchedliterature: Sahoo et al., Synthesis, 2003, 346

Reference Example 16: Preparation of N-Phenyldibromomaleimide

Aniline (72 μL, 0.788 mmol) was added to a solution of dibromomaleicanhydride (200 mg, 0.788 mmol) in AcOH (10 mL). The mixture was stirredfor 3 h at RT and at 130° C. for 90 mins. After cooling, the mixture wasconcentrated to dryness and traces of AcOH removed by azeotrope withtoluene. The tan residue was purified using silica flash chromatography(5% EtOAc/95% petroleum ether) to yield the desired compound as a paleyellow solid (166 mg, 60%), δ_(H) (600 MHz, CDCl₃) 7.48 (m, 2H, ArH),7.41 (tt, 1H, J=7.4 and 1.1 Hz, ArH), 7.33 (m, 2H, ArH); δ_(C) (150 MHz,CDCl₃) 163.0, 131.0, 130.0, 129.5, 128.8, 126.2.

Reference Example 17: Preparation of 3,4-Diiodo-pyrrole-2,5-dione

To dibromomaleimide (500.0 mg, 2.0 mmol) in acetic acid (50 ml) wasadded sodium iodide (886.5 mg, 5.9 mmol). The reaction mixture washeated to 120° (C and refluxed for 2 h. The reaction was allowed to cooldown to RT. H₂O (50 ml) was added and kept at 4° C. for 15 h. The yellowprecipitate was filtered off and air dried to afford the desiredcompound as an orange crystalline powder (415 mg, 60%). ¹H NMR (500 MHz,MeOD): no signals; ¹³C NMR (125 MHz, MeOD): δ=169.3 (C), 119.5 (C); IR(solid, cm⁻¹): 3244 (s), 2944 (m), 2833 (m); MS (EI) m/z, (%): 349 (M,83), 179 (100); Mass calc. for C₄H₁₂O₂N: 348.80912. Found: 348.81026,m.p. 238-241° C. (Literature: 254-255° C.).

Reference Example 18: Preparation of3,4-Bis-(2-hydroxy-ethylsulfanyl)-pyrrole-2,5-dione

To 2-mercaptoethanol (683.8 μl, 9.8 mmol) in buffer (10) ml, 150 mMNaCl, 100 mM sodium phosphate, pH 8.0, 5.0% DMF) was addeddi-bromonmaleinmide (1 g, 3.9 mmol) in DMF (2.5 ml, final concentrationDMF 7.5%). The reaction was stirred for 30 min at RT and lithiumchloride (20 g) was added. The aqueous reaction mixture was extractedwith ethyl acetate (7×150 ml). The organic layers were combined, thesolvent removed in vacuo and the residual material was purified by flashchromatography on silica gel (petroleum ether:ethyl acetate, gradientelution from 1:1 to 1:9). Fractions containing the product werecollected and the solvent were removed in vacuo. The still impureproduct was purified by flash chromatography on silica gel(methanol:dichloromethane, gradient elution from 0.5-10.0% methanol) toafford the desired compound as a yellow solid (518 mg, 53%). λ_(max) (50mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF)/318 nm (ε/dm³ mol⁻¹cm⁻¹ 1855); ¹H NMR (500 MHz, MeOD): δ=3.74 (t, 4H, J=6.4, 2×HO—CH₂),3.41 (t, 4H, J=6.3, 2×S—CH₂) ¹³C NMR (125 MHz, MeOD): δ=168.5 (C), 137.2(C), 62.3 (CH₂), 34.4 (CH₂); IR (solid, cm⁻¹): 3344 (s), 2500 (m), 2078(w); MS (EI) m/z, (%): 250 (M, 43), 232 (100), 161 (37); Mass calc. forC₈H₁₁O₄NS₂: 250.02077. Found: 250.02126; m.p. 46-50° C.

Reference Example 19: Preparation of3,4-Bis-phenylsulfanyl-pyrrole-2,5-dione

To dibromomaleimide (80.0 mg, 0.3 mmol) and sodium hydrogencarbonate(130.2 mg, 1.6 mmol) in methanol (6 ml) was slowly added benzenethiol(66.6 μl, 0.7 mmol) in methanol (1 ml). The reaction was stirred for 15min at RT. The solvent was removed in vacuo and the residual materialwas purified by flash chromatography on silica gel (petroleumether:ethyl acetate, gradient elution from 9:1 to 7:3) to afford thedesired product as bright yellow crystals (73 mg, 75%). λ_(max) (50 mMsodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF)/412 nm (ε/dm³ mol⁻¹ cm⁻¹2245); ¹H NMR (500 MHz, MeOD): δ=7.27-7.22 (m, 6H, Ar—H), 7.16-7.14 (m,4H, Ar—H); ¹³C NMR (125 MHz, MeOD): δ=169.3 (C), 137.6 (C), 135.4 (C),132.4 (CH), 130.1 (CH), 129.1 (CH); IR (solid, cm⁻¹): 3285 (m), 3059(w), 2924 (w), 1774 (m), 1715 (s); MS (CH) m/z, (%): 314 (M+H, 100), 206(13), 111 (12); Mass calc. for C₁₆H₁₁O₂NS₂[+H]: 314.0231. Found:314.0309; m.p. 102-104° C. (Literature: 123-126° C.).

Reference Example 20: Preparation of3,4-Bis-(pyridine-2-ylsulfanyl)-pyrrole-2,5-dione

To dibromomaleimide (300.0 mg, 1.2 mmol) and sodium acetate (480.0 mg,5.9 mmol) in methanol (15 ml) was slowly added 1H-pyridine-2-thione(275.8 mg, 2.5 mmol) in methanol (4 ml). The reaction was stirred for 15min at RT. The solvent was removed in vacuo and the residual materialwas purified by flash chromatography on silica gel(methanol:dichloromethane, gradient elution from 0.5-3.0%) to afford thedesired product as a dark yellow powder (190 mg, 51%). λ_(max) (50 mMsodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF)/395 nm (d dm³ mol⁻¹ cm⁻¹3508); ¹H NMR (500 MHz, MeOD): δ=8.37 (d, 2H, J=3.8, 2×N—CH), 7.70 (t,2H, J=6.9, 2×C—CH—CH), 7.38 (d, 2H, J=7.9, 2×C—CH), 7.26 (t, 2H, J=6.5,2×N—CH—CH); ¹³C NMR (125 MHz, MeOD): δ=168.5 (C), 154.7 (C), 150.9 (CH),140.0 (C), 139.0 (CH), 126.8 (CH), 123.7 (CH); IR (solid, cm⁻¹): 2926(m), 2734 (w), 1771 (w), 1726 (s), 1619 (m); MS (CI) m/z, (%): 316 (M+H,5), 152 (10), 126 (34), 112 (100); Mass calc. for C₁₄H₉O₂N₃S₂[+H]:316.0214. Found: 316.0223; m.p. 70-72° C.

Reference Example 21: Preparation of N-PEG300 dibromomaleimide

The reaction was carried out under strictly dry conditions. Totriphenylphosphine (193.9 mg, 0.7 mmol) in THF (5 mL) was addeddrop-wise diisopropyl azodicarboxylate (145.6 μl, 0.7 mmol) at −78° C.The reaction was stirred for 5 min and PEG300 (200.0 mg, 0.6 mmol) inTHF (4 mL) was added drop-wise. The reaction was stirred for 5 min andneopentyl alcohol (45.8 mg, 0.5 mmol) in THF (1 ml) was added. Thereaction was stirred for 5 min and 3,4-dibromomaleimide (189.4 mg, 0.7mmol) in THF (2 ml) was added. The reaction was stirred for 10 min. thecold bath removed and stirred for 20 h at ambient temperature. Thesolvent was removed in vacuo and the residual material was purified byflash chromatography on silica gel (methanol:dichloromethane, gradientelution from 0.5-5.0% methanol). Fractions containing the product werecollected and the solvent was removed in vacuo. The still impure productwas purified by flash chromatography on silica gel (petroleumether:ethyl acetate, gradient elution from 7:3 to 2:8) to afford thedesired compound as a yellow oil (137 mg, 40%) with 97.5% purity. ¹H NMR(500 MHz, CDCl₃): δ=3.76 (t, 2H, J=5.6, N—CH₂), 3.64-3.52 (m, 24H,12×CH₂—O), 3.49 (t, 2H, J=4.4, N—CH₂—CH₂), 3.32 (s, 3H, O—CH₃); ¹³C NMR(125 MHz, CDCl₃): δ=163.8 (2×C), 129.5 (2×C), 72.0 (CH₂), 70.7-70.5(9×CH₂), 70.1 (2×CH₂), 67.5 (CH₂), 59.1 (CH₃), 39.0 (CH₂); IR (solid,cm⁻¹): 3496 (w), 2869 (m), 1786 (m), 1720 (s), 1594 (m); MS (CI) m/z,(%): 580 (⁸¹M+H, 12), 578 (^(81.79)M+H, 23), 576 (⁷⁹M+H, 12), 279 (100),84 (61); Mass calc. for C₁₉H₃₁ ⁷⁹Br₂O₉N[+H]: 576.0444. Found: 576.0437.

Reference Example 22: Preparation of N-PEG5000 Dibromomaleimide

The reaction was carried out under strictly dry conditions. Totriphenylphosphine (154.6 mg, 0.6 mmol) in a mixture of THF (8 mL) andDCM (3 mL) was added drop-wise diisopropyl azodicarboxylate (116.0 μl,0.6 mmol) at −78° C. The reaction was stirred for 5 min and PEG5000(2950.0 mg, 0.6 mmol) in dichloromethane (7 mL) was added drop-wise. Thereaction was stirred for 5 min and neopentyl alcohol (26.5 mg, 0.3 mmol)in a mixture of THF (1 ml) and DCM (1 ml) was added. The reaction wasstirred for 5 min and 3,4-dibromomaleimide (150.0 mg, 0.6 mmol) in THF(2 ml) was added. The reaction was stirred for 5 min, the cold bathremoved and stirred for 20 h at ambient temperature. The solvent wasremoved in vacuo and the residual material was purified by flashchromatography on silica gel (methanol:dichloromethane, gradient elutionfrom 0.5-5.0% methanol). Fractions containing the product were collectedand the solvent was removed in vacuo. The still impure product waspurified by very slow flash chromatography on silica gel(methanol:dichloromethane, gradient elution from 0.5-6.0% methanol) toafford desired compound as a pale green crystalline powder (417 mg,13%). ¹H NMR (500 MHz, CDCl₃): δ=3.58 (s, 4×n H, CH₂); ¹³C NMR (125 MHz,CDCl₃): δ=163.8 (C), 129.5 (C), 70.6 (CH₂); IR (solid, cm⁻¹): 3517 (w),2872 (s), 1977 (w), 1727 (m), 1641 (w); m.p. 51-55° C.

Reference Example 23: Preparation of N-PEG5000 Dithiophenolmaleimide

The reaction was carried out under strictly dry conditions. Totriphenylphosphine (167.7 mg, 0.6 mmol) in a mixture of THF (8 ml) andDCM (3 ml) was added drop-wise diisopropyl azodicarboxylate (125.9 μl,0.6 mmol) at −78° C. The reaction was stirred for 5 min and PEG5000(1600.0 mg, 0.3 mmol) in DCM (7 ml) was added drop-wise. The reactionwas stirred for 5 min and neopentyl alcohol (56.3 mg, 0.6 mmol) in amixture of THF (1 ml) and DCM (1 ml) was added. The reaction was stirredfor 5 min and 3,4-dithiophenolmaleimide (200.0 mg, 0.6 mmol) in THF (3ml) was added. The reaction was stirred for 5 min, the cold bath removedand stirred for 20 h at ambient temperature. The solvent was removed invacuo and the residual material was purified by flash chromatography onsilica gel (methanol:dichloromethane, gradient elution from 0.5-10.0%methanol). Fractions containing the product were collected and thesolvent was removed in vacuo. The still impure product was purified byflash chromatography on TLC grade silica gel (methanol:dichlormethane,gradient elution from 0.0-10.0% methanol) to afford the desired compoundas a bright yellow crystalline powder (1.24 g, 73%). ¹H NMR (500 MHz,CDCl₃): δ=7.26 (dd, H, J=7.7, J=4.5, CH), 7.23 (dd, 2H, J=8.4, J=6.6,CH), 7.19 (dd, 2H, J=8.4, J=6.8, CH), 3.63 (s, 4×n H, CH₂); ¹³C NMR (125MHz, CDCl₃): δ=166.7 (C), 135.7 (C), 131.9 (CH), 129.1 (C), 129.0 (CH),128.4 (CH), 70.6 (CH₂); IR (solid, cm⁻¹): 3498 (w), 2881 (s), 1959 (w),1711 (m); m.p. 57-59° C.

Reference Example 24: Preparation of 2,3-dibromo-maleic anhydride

Under an inert atmosphere, a solution of maleic anhydride (1.50 g, 15.3mmol, 1 eq), aluminium trichloride (300 mg, 0.21 mmol, cat.) and bromine(4.95 g, 30.6 mmol, 2 eq) was heated at 160° C. in a sealed ampule(note—blast shield) for 16 h. Upon cooling to 21° C. the reactionmixture was stirred for a further 24 h and carefully opened to air.EtOAc was added and the solid filtered off and repeatedly washed withfurther EtOAc. The filtrate was finally concentrated in vacuo to givethe title compound was a yellow solid which was used without furtherpurification (3.05 g, 11.9 mmol, 78% yield), m.p 107-110° C.; ¹³C NMR(150 MHz, CD₃OD) δ 163.33 (s), 125.28 (s); IR (MeOH) 1769, 1706, 1590cm⁻¹; HRMS (CI) calcd for C₄O₃Br₂ [M]⁺ 253.82087, 253.82082 observed.

Reference Example 25: Preparation of tert-ButylN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate

A solution of di-tert-butyl-dicarbonate (1.10 g, 5.0 mmol, 1 eq) inCH₂Cl₂ (5 mL) was added dropwise to a solution of2-[2-(2-aminoethoxy)ethoxy]ethanamine (7.32 mL, 50.0 mmol, 10 eq) inCH₂Cl₂ (15 mL). The resulting reaction mixture was stirred at 21° C. for24 h. The CH₂Cl₂ was then removed in vacuo to leave a colourlessresidue. Addition of EtOAc (125 mL) caused formation of a whiteprecipitate, which was washed with a saturated solution of Na₂CO₃ (3×50mL), dried over MgSO₄, and concentrated in vacuo. Further purificationby column chromatography (8:2 CH₂Cl₂/MeOH) furnished the desiredmonoprotected amine as a colourless oil (0.69 g, 2.80 mmol, 56% yield).¹H NMR (500 MHz, CDCl₃) δ 5.27 (bs, 1H, NH), 3.54-3.52 (m, 4H, OCH₂),3.47-3.42 (m, 4H, OCH₂), 3.23-3.22 (m, 2H, NCH₂), 2.80 (t, J=5.0, 2H,NCH₂), 2.05 (bs, 2H, NH), 1.35 (s, 9H, CH₃); ¹³C NMR (125 MHz, CDCl₃) δ156.08 (s), 79.09 (s), 73.19 (t), 70.21 (t), 70.16 (t), 41.59 (t), 40.32(t), 28.40 (q), * 1 t missing; IR (neat) 3344, 2869, 1692 cm⁻¹; HRMS(CI) calcd for C₁₁H₂₅N₂O₄ [M+H]⁺ 249.18143, observed 249.18251.

Reference Example 26: Preparation oftert-Butyl-N-(2-(2-(2-(5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanoylamino)ethoxy)ethoxy)ethyl)carbamate

A solution of biotin (0.59 g, 2.42 mmol, 1.5 eq), HBTU (0.79 g, 2.10mmol, 1.3 eq) and DIEA (0.45 mL, 2.60 mmol, 1.6 eq) in DMF (15 mL) wasstirred for 20 min at 21° C. before being added dropwise to a solutionof tert-butyl-N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (400 mg,1.61 mmol, 1 eq) in DMF (10 mL). The reaction mixture was stirred for 2h at 21° C., after which the DMF was removed in vacuo to give a yellowresidue.

The crude product was purified by column chromatography (gradient 2-10%MeOH/CH₂Cl₂) to yield the desired compound as a white solid (0.61 g,1.29 mmol, 80% yield), m.p. 106-108° C.; [α]_(D) ^(20.0) +23.0 (c 0.6,CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 4.55 (dd, J=5.0, 7.5 Hz, 1H,NHC(O)NHCH), 4.36 (dd, J=5.0, 7.5 Hz, 1H, NHC(O)NHCH), 3.62 (bs, 6H,OCH₂), 3.59-3.55 (m, 2H, OCH₂), 3.46 (m, 2H, NCH₂), 3.31 (m, 2H, NCH₂),3.17 (dt, 3.0, 5.0 Hz, 1H, SCH), 2.92 (dd, J=5.0, 13.0 Hz, 1H, SCHH),2.79 (d, J=13.0 Hz, 1H, SCHH), 2.27 (t, J=7.0 Hz, 2H, NHC(O)CH₂CH₂CH₂),1.71 (m, 4H, NHC(O)CH₂CH₂CH₂CH₂), 1.47 (br, 11H, C(CH₃)₃ &NHC(O)CH₂CH₂CH₂CH₂); ¹³C NMR (125 MHz, CDCl₃) δ 173.69 (s), 163.92 (s),155.99 (s), 79.14 (s), 70.03 (t), 69.69 (br t), 61.58 (d), 60.06 (d),55.19 (d), 40.16 (t), 39.96 (t), 38.91 (t), 35.44 (t), 28.09 (q), 27.80(t), 27.67 (t), 25.23 (t), * 2 t absent; IR (neat) 3307, 2933, 1691cm⁻¹; HRMS (ES) calcd for C₂₁H₃₈N₄O₆NaS [M+Na]⁺ 497.2410, observed497.2423.

Reference Example 27: Preparation of2-(2-(2-(5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanoylamino)ethoxy)ethoxy)ethylammonium;2,2,2-trifluoroacetate

A solution of tert-butylN-(2-(2-(2-(5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanoylamino)ethoxy)ethoxy)ethyl)carbamate(0.61 g, 1.29 mmol) in CH₂Cl₂ (5 mL) and TFA (5 mL) was stirred at 21°C. for 24 h. Toluene was then added (×2) and the solvent removed invacuo to yield the desired compound as an oil (0.63 g, 1.29 mmol, 100%yield). [α]_(D) ^(20.0) +41.0 (c 0.49, MeOH); ¹H NMR (400 MHz, CD₃OD) δ4.53 (dd, J=5.0, 7.5 Hz, 1H, NHC(O)NHCH), 4.33 (dd, J=5.0, 7.5 Hz, 1H,NHC(O)NHCH), 3.71 (t, J=5.0 Hz, 2H, OCH₂CH₂NH₃), 3.65 (br, 4H, OCH₂),3.57 (t, J=5.0 Hz, 2H, OCH₂), 3.38 (t, J=5.0 Hz, 2H, OCH₂), 3.22 (dt,J=5.0, 8.5 Hz, 1H, SCH), 3.13 (t, J=5.0 Hz, 2H, C(O)NHCH₂CH₂O), 2.94(dd, J=5.0, 13.0 Hz, 1H, SCHH), 2.74 (d, J=13.0 Hz, 1H, SCHH), 2.24 (t,J=7.5 Hz, 2H, NHC(O)CH₂CH₂CH₂), 1.76-1.43 (m, 6H, NHC(O)CH₂CH₂CH₂CH₂);¹³C NMR (100 MHz, CD₃OD) δ 174.98 (s), 164.76 (s), 69.92 (t), 69.83 (t),69.22 (t), 66.46 (t), 62.08 (d), 60.36 (d), 55.59 (d), 39.65 (t), 39.24(t), 38.77 (t), 35.29 (t), 28.29 (t), 28.06 (t), 25.44 (t); IR (MeOH)3300, 2941, 1686 cm⁻¹; HRMS (ES) calcd for C₁₆H₃₁N₄O₄S [M+H]⁺ 375.2066,observed 375.2060.

Reference Example 28: Preparation ofN-(2-(2-(2-(3-bromo-2,5-dioxo-pyrrol-1-yl)ethoxy)ethoxy)ethyl)-5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanamide

Monobromomaleic anhydride (45.0 mg, 0.25 mmol, 1 eq) was added in oneportion to a solution of2-(2-(2-(5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanoylamino)ethoxy)ethoxy)ethylammonium2,2,2-trifluoroacetate (124 mg, 0.25 mmol, 1 eq) in AcOH (10 mL) and thereaction mixture heated to 170° C. for 3 h. Upon cooling to 21° C.toluene was added and the AcOH azeotropically removed in vacuo (×2) togive crude product. Column chromatography (gradient 2-10% MeOH/CH₂Cl₂)yielded the desired compound as a white solid (70.0 mg, 0.13 mmol, 52%yield), m.p. 95-98° C.; [α]_(D) ^(20.0) +65.1 (c 0.15, MeOH): ¹H NMR(600 MHz, CD₃OD) δ 7.17 (s, 1H, CHCBr), 4.51 (dd, J=5.0, 8.0 Hz, 1H,NHC(O)NHCH), 4.33 (dd, J=5.0, 8.0 Hz, 1H, NHC(O)NHCH), 3.77 (t, J=5.5Hz, 2H, OCH₂), 3.68 (t, J=5.5 Hz, 2H, OCH₂), 3.63 (m, 2H, OCH₂), 3.58(m, 2H, OCH₂), 3.53 (t, J=5.5 Hz, 2H, NCH₂), 3.37 (t, J=5.5 Hz, 2H,NCH₂), 3.24 (td, J=5.0, 8.0 Hz, 1H, SCH), 2.95 (dd, J=5.0, 12.5 Hz, 1H,SCHH), 2.73 (d, J=12.5 Hz, 1H, SCHH), 2.26 (t, J=7.0 Hz, 2H,NHC(O)CH₂CH₂CH₂), 1.69 (m, 4H, CH₂CH₂CH₂), 1.47 (quintet, J=7.0 Hz, 2H,CH₂CH₂CH₂); ¹³C NMR (150 MHz, CD₃OD) δ 176.12 (s), 170.13 (s), 166.97(s), 166.08 (s), 133.63 (s), 132.05 (d), 71.22 (t), 71.11 (t), 70.61(t), 68.69 (t), 63.35 (d), 61.61 (d), 57.03 (d), 41.09 (t), 40.31 (t),39.09 (t), 36.75 (t), 29.78 (t), 29.50 (t), 26.87 (t); IR (MeOH) 3355,2970, 1737 cm⁻¹; HRMS (ES) calcd for C₂₀H₂₉N₄O₆ NaSBr [M+Na]⁺ 555.0889,observed 555.0905.

Reference Example 29: Preparation ofN-(2-(2-(2-(3,4-dibromo-2,5-dioxo-pyrrol-1-yl)ethoxy)ethoxy)ethyl)-5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanamide

Dibromomaleic anhydride (108 mg, 0.42 mmol, 1 eq) was added in oneportion to a solution of2-(2-(2-(5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanoylamino)ethoxy)ethoxy)ethylammonium2,2,2-trifluoroacetate (205 mg, 0.42 mmol, 1 eq) in AcOH (10 mL) and thereaction mixture heated to 170° C. for 2 h. Upon cooling to 21° C.toluene was added and the AcOH azeotropically removed in vacuo (×2) togive crude product. Column chromatography (gradient 2-7% MeOH/CH₂Cl₂)yielded the desired compound as a white solid (123 mg, 0.20 mmol, 48%yield), m.p. 100-102° C.; [α]_(D) ^(20.0) +71.0 (c 0.15, MeOH); ¹H NMR(600 MHz, CD₃OD) δ 4.53 (dd, J=5.0, 8.0 Hz, 1H, NHC(O)NHCH), 4.34 (dd,J=5.0, 8.0 Hz, 1H, NHC(O)NHCH), 3.82 (t, J=5.5 Hz, 2H, OCH₂), 3.70 (t,J=5.5 Hz, 2H, OCH₂), 3.63 (m, 2H, OCH₂), 3.59 (m, 2H, OCH₂), 3.53 (t,J=5.5 Hz, 2H, NCH₂), 3.37 (t, J=5.5 Hz, 2H, NCH₂), 3.24 (dt, J=5.0, 8.0Hz, 1H, SCH), 2.96 (dd, J=5.0, 13.0 Hz, 1H, SCHH), 2.73 (d, J=13.0 Hz,1H, SCHH), 2.26 (t, J=7.5 Hz, 2H, NHC(O)CH₂CH₂CH₂), 1.74 (m, 4H,CH₂CH₂CH₂), 1.49 (quintet, J=7.5 Hz, 2H, CH₂CH₂CH₂); ¹³C NMR (150 MHz,CD₃OD) δ 174.83 (s), 164.71 (s), 164.06 (s), 129.00 (s), 69.80 (t),69.72 (t), 69.24 (t), 67.19 (t), 61.97 (d), 60.22 (d), 55.64 (d), 39.67(t), 39.03 (t), 38.56 (t), 35.42 (t), 28.39 (t), 28.11 (t), 25.47 (t);IR (MeOH) 2970, 1724, 1365, 1217 cm⁻¹; HRMS (ES) calcd forC₂₀H₂₈N₄O₆NaSBr₂ [M+Na]⁺ 631.9916, observed 631.9937.

Reference Example 30: Preparation of N-Fluorescein Bromomaleimide

Dibronomaleic anhydride (346 mg, 1.95 mmol) was added in one portion toa solution of fluoresceinamine isomer 1 (678 mg, 1.95 mmol) in aceticacid (65 mL) and the reaction mixture was stirred for 12 hours at roomtemperature in a sealed tube. The reaction mixture was then heated to150° C. for 3 h. Upon cooling to room temperature the solid was filteredand dried (toluene azeotrope) to afford the desired compound as anorange solid (722 mg, 1.43 mmol, 73% yield). ¹H NMR (600 MHz, DMSO) δ7.99 (d, 1H, J=1.7, 1H, H-11), 7.77 (dd, 1H, J=1.9 and 8.2, 1H, H-7),7.73 (s, 1H, H-3), 7.43 (d, J=8.2, 1H, H-8), 6.69 (nm, 6H, 2×H-16,2×H-17, 2×H-18); ¹³C NMR (175 MHz, DMSO) δ 167.93 (C═O), 167.63 (C═O),164.48 (C═O), 159.62 (2×C18), 151.79 (2×C20), 151.52 (C6), 133.68 (C7),133.02 (Ar), 132.90 (C3), 131.23 (C), 129.15 (2×Ar—H), 126.73 (C),124.82 (C11), 122.29 (C8), 112.77 (2×Ar—H), 109.08 (2×Ar), 102.30(2×Ar—H), 83.36 (C14); IR (solid, cm⁻¹) 3064 (w), 1726 (s); MS (ES+)nm/z (relative intensity): 508 ([⁸¹M], 95), 506 ([⁷⁹M], 100); Exact masscalcd for [C₂₄H₁₃O₇N⁷⁹Br] requires 505.9875. Found 505.9833 (ES+).

Reference Example 31: Preparation of N-Fluorescein Dibromomaleimide

Dibromomaleic anhydride (77.0 mg, 0.30 mmol) was added in one portion toa solution of fluoresceinamine isomer 1 (105 mg, 0.30 mmol) in aceticacid (10 mL) and the reaction mixture was stirred for 6 h at roomtemperature. The solid was then filtered off, washed with ethyl acetate,and redissolved in acetic acid (10 mL). The reaction mixture was thenheated to reflux for 3 h. Upon cooling to room temperature toluene (10ml) was added and the solvent removed in vacuo, affording the desiredcompound as an orange solid (148 mg, 0.25 mmol, 84% yield), δ ¹H NMR(400 MHz, CD₃OD) δ 8.07 (d, 1H, J=1.5, H-11), 7.81 (dd, 1H, J=1.5 and8.0, H-7), 7.34 (d, 1H, J=8.5, H-8), 6.71-6.58 (m, 6H, 6×Ar—H); ¹³C NMR(100 MHz, CD₃OD) δ 170.23 (C═O), 164.34 (2×C═O), 161.63 (2×C), 154.18(2×C), 152.93 (C), 134.59 (C), 134.19 (Ar—H), 131.01 (C), 130.35 (Ar—H),129.25 (2×C), 126.25 (2×Ar—H), 123.63 (Ar—H), 113.84 (2×Ar—H), 111.02(2×C), 103.55 (2×Ar—H); IR (solid, cm⁻¹) 3064 (w), 1732 (s); MS (ES+)m/z, (relative intensity): 586 ([⁸¹⁺⁸¹M], 30), 584 ([⁷⁹⁺⁸¹M], 100), 582([⁷⁹⁺⁷⁹M], 100); Exact mass calcd for [C₂₄H₁₀O₇N⁷⁹Br₂] requires581.8824. Found 581.8824 (ES+).

Reference Example 32: Preparation of Tert-butyl 2-aminomethylcarbamate

Di-tertbutyldicarbonate (3.26 g, 15 mmol, 1 eq) in DCM (30 mL) wasadded, dropwise, to a solution of ethylenediamine (10 ml, 150 mmol, 10eq) in DCM (30 mL) under an argon atmosphere over two hours using anautoinjecter. Based on TLC analysis (eluent: 90% EtOAc: 10% MeOHR_(f(8))=0.23) the reaction reached completion 30 minutes after the endof the addition. The DCM was removed under reduced pressure using aBüchi. The resultant residue was taken up in EtOAc (40 mL) and washedwith saturated Na₂CO₃ (3×20 mL), dried over MgSO4, and concentrated invacuo to obtain the desired product (2.08 g; 12.98 mmol, 87%) as a whitefoam. mp (104-106° C.), δ_(H) ¹H NMR (300 MHz CDCl₃): 4.95 (broadsinglet, 1H, NH), 3.12 (q, J=6.4 Hz, 2H, CH₂), 2.78 (t, J=5.9 Hz, 2H,CH₂), 1.42 (s, 9H, 3CH₃), 13C NMR (CDCl₃): 28.06, 41.51, 43.02, 78.82,155.9 IR: 3354.9 cm⁻¹, [M+H]⁺: 161.00

Reference Example 33: Preparation of tert-butyl2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethylcarbamate

A round bottom flask was flame dried and equipped with a stirrer bar anda solution of amine (0.57 g, 3.6 mmol, 1 eq) in dry DCM (150 mL) underan argon atmosphere. Dansyl chloride (1.05 g, 3.92 mmol, 1.1 eq) in dryDCM (150 mL) and triethylamine (1.3 ml, 9.29 mmol, 2.5 eq) were addedthrough a septum in one portion. Reaction was monitored by TLC (eluent:35% EtOAc: 65% Petroleum ether R_(f(9))=0.27, fluorescent green underlong UV), the reaction was complete after 4 hours. Followingpurification by column chromatography (eluent: 35% EtOAc: 65% Petroleumether), the desired compound was formed (1.24 g, 3.15 mmol, 88%) as asticky, clear green oil. δ_(H) ¹H NMR (CDCl3): 8.55 (d, J=8.55 Hz, 1H,CH), 8.46 (d, J=8.51 Hz, 1H, CH), 8.33 (d, J=8.67 Hz, 1H, CH), 7.57 (m,2H, 2×CH), 7.26 (d, J=7.08 Hz, 1H, CH), 3.07 (quartet, J=6.58 Hz, 2H,CH₂), 2.89 (m, 2H, CH₂), 2.85 (s, 6H, 2×CH₃), 1.35 (s, 9H, 3×CH₃), 13CNMR (CDCl3): 158.01, 153.05, 136.5, 131.49, 131.09, 130.02, 129.54,124.48, 120.59, 116.41, 80.41, 45.9, 43.81, 41.5, 28.5. MS: [M+H]+:393.16.

Reference Example 34: Preparation of5-(3-aminopropylsulfonyl)-N,N-dimethylnaphthalen-1-amine2,2,2-trifluoroacetate

To a flask containing BOC-carbamate (1.24 g, 3.15 mmol), TFA (40 ml) wasadded in one portion. The resulting grey solution was stirred for 2hours at room temperature (ca. 25° C.). Upon completion the solution wasconcentrated in vacuo and azeotroped with tolune (5×10 ml). Theresultant crude product was then purified by column chromatography(eluent: EtOAc 1:2 Petroleum ether R_(f(10))=0.20). After concentratingthe relevant fractions in vacuo, to the yellow oil that resulted, DCM(100 ml) was added and the solution placed in an ice bath for 2 hours,this solution was fluorescent under long-wave UV. The desired compound(1.25 g, 3.10 mmol, 97%) crashed out of solution as a white solid and itwas filtered off and washed with diethyl ether under gravity. Mp(114-116° C.); δ_(H) ¹H NMR (500 MHz MeOD): 8.64 (d, J=8.3 Hz, 1H, CH),8.35 (d, J=8.45 Hz, 1H, CH), 8.32 (d, J=8.65 Hz, 1H, CH), 7.67 (m, 2H,2×CH), 7.34 (d, J=7.3 Hz, 1H, CH), 3.03 (quartet, 4H, 2×CH₂), 2.84 (s,6H, 2×CH₃), δ_(C) ¹³C NMR (500 MHz MeOD): 153.41, 135.78, 131.69,131.32, 130.81, 130.56, 129.48, 124.29, 120.07, 116.635, 66.91, 45.79,41.27, 40.78, 15.45. ¹⁹F NMR (300 MHz CDCl₃); −76.89; IR: 3092 cm⁻¹,2901.5 cm⁻¹, MS: [M+H]+: 294

Reference Example 35: Preparation of(E)-2-bromo-4-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethylamino)-4-oxobut-2-enoicAcid

An oven-dried 500 ml round bottomed flask was equipped with a stirringbar. Amine salt (1.09 g) was dissolved in 25 ml acetic acid and added tothe flask. To the resulting light yellow solution, Bromomaleic anhydridewas added and reaction was monitored by TLC (eluent; 10% methanol: 90%EtOAc, R_(f(11))=0.7). After 1.5 hours of stirring at room temperature(25° C.) the acetic acid was removed in vacuo. The desired compound wasused without further purification. 1H NMR (500 Mz CDCl₃ (Crude)): δ_(H)8.6 (d, J=8.56 MHz, 1H, CH), 8.35 (d, 1H, J=8.27 Hz, CH), 8.22 (d, 1H,J=8.57 Hz, CH), 7.64 (m, 2H, 2×CH), 7.30 (d, J=7.60 Hz, 1H, CH), 5.48(s, 1H, CH)/5.03 (s, 1H, CH), 3.00 (m, 4H, 2×CH₂), 2.88 (s, 9H, 2×CH₃)

Reference Example 36: Preparation ofN-(2-(3-bromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-5-(dimethylamino)naphthalene-1-sulfonamide

The acid was dissolved in acetic acid (25 mL) and loaded into an ovendried 500 ml round bottom flask. A condenser was fitted and the reactionwas placed under reflux (170° C.) for 2 hours. The acetic acid was thenremoved from the crude mixture in vacuo and the resultant oil wasaziotroped with toluene (5×10 ml). The resultant oil was purified bycolumn chromatography (eluent: 30% ethylacetate: 70% petroleum ether,R_(f(12))=0.2 in the aforementioned eluent system). Once the very slowcolumn was completed, the more mobile fraction was collected and thesolvent removed. The resultant brown oil was left to stand in neat ethylacetate (50 ml) for 1 hour in an ice bath. The desired product (0.961 g,80%) crashed out of solution as a brown solid (powder like texture),this was filtered under gravity and washed with diethyl ether (20 ml).mp (166-170° C.); ¹H NMR (600 MHz DMSO): δ_(H) 8.53 (d, J=8.46 Hz, 1H,CH), 8.21 (d, 1H, J=8.40 Hz, CH), 8.17 (d, 1H, J=−8.58 Hz, CH), 7.56 (m,2H, 2×CH), 7.18 (d, J=7.50 Hz, 1H, CH), 6.46 (s, 1H, maleimide olefinC—H), 5.11 (t, J=6.24, 1H, NH), 3.56 (m, 2H, CH₂), 3.2 (m, 2H, CH₂),3.91 (s, 6H, 2×CH₃). δ_(C) ¹³C NMR (600 MHz DMSO): 168.62, 165.33,151.38, 151.38, 135.62, 132.33, 130.13, 129.60, 129.09, 128.85, 128.30,127.96, 123.61, 118.99, 115.22, 45.11, 40.05, 39.37, 38.47.

Reference Example 37: Preparation of4-Bromo-1,2-diethyl-1,2-dihydro-pyridazine-3,6-dione (BrDDPD)

A mixture of monobromomaleic anhydride (177 mg, 1.0 mmol) andN,N′-diethylhydrazine (88 mg, 1.0 mmol) in glacial AcOH (3 mL) washeated at 130° C. for 16 h. The solvent was removed in vacuo and thecrude residue purified by column chromatography (neat CH₂Cl₂-5%MeOH/CH₂Cl₂) to give4-bromo-1,2-diethyl-1,2-dihydro-pyridazine-3,6-dione as a yellow solid(159 mg, 0.64 mmol, 64%): ¹H NMR (600 MHz, CDCl₃) δ 7.31 (s, 1H), 4.14(q, J=7.0 Hz, 2H), 4.07 (q, J=7.0 Hz, 2H), 1.26 (t, J=7.0 Hz, 3H), 1.22(t, J=7.0 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 156.2 (s), 154.3 (s),136.0 (d), 133.7 (s), 41.9 (t), 40.7 (t), 13.3 (q), 13.3 (q); IR (solid)3058, 2979, 2938, 1631, 1595 cm⁻¹; LRMS (CI) 249 (100, [M⁸¹Br+H]⁺), 247(100, [M⁷⁹Br+H]⁺); HRMS (CI) calcd for C₈H₁₂BrN₂O₂[M+H]⁺ 249.0082,observed 249.0086.

Reference Example 38: Preparation of4,5-Dibromo-1,2-diethyl-1,2-dihydro-pyridazine-3,6-dione (DiBrDDPD)

A mixture of dibromomaleic anhydride (256 mg, 1.0 mmol) andN,N′-diethylhydrazine (88 mg, 1.0 mmol) in glacial AcOH (3 mL) washeated at 130° C. for 16 h. The solvent was removed in vacuo and thecrude residue purified by column chromatography (neat CH₂Cl₂-5%MeOH/CH₂Cl₂) to give4,5-dibromo-1,2-diethyl-1,2-dihydro-pyridazine-3,6-dione as a yellowsolid (202 mg, 0.62 mmol, 62%): ¹H NMR (600 MHz, CDCl₃) δ 4.17 (q, J=7.0Hz, 4H), 1.28 (t, J=7.0 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 153.3 (s),136.1 (s), 42.4 (t), 13.2 (q); IR (solid) 2979, 2937, 1630, 1574 cm⁻¹;LRMS (EI) 328 (50, [M⁸¹Br⁸¹Br]⁺⁻), 326 (100, [M⁸¹Br⁷⁹Br]⁺⁻), 324 (50,[M⁷⁹Br⁷⁹Br]⁺⁻): HRMS (EI) calcd for C₈H₁₀Br₂N₂O₂[M⁷⁹Br⁷⁹Br]⁺⁻ 323.9104,observed 323.9097.

Reference Example 39: Preparation of N-Boc-Cys(Mal)-OMe

To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.15 mmol) and sodiumacetate (13 mg, 0.15 mmol) in methanol (3 mL) was added bromomaleimide(30 mg, 0.17 mmol) in methanol (3 mL). After 1 minute solvent wasremoved in vacuo. Purification by flash chromatography (gradient elutionin 50% ethyl acetate in petroleum ether to ethyl acetate) afforded apale yellow powder N-Boc-Cys(Mal)-OMe (51 mg, 0.15 mmol) in 100%. δ_(H)(500 MHz, CDCl₃) 7.63 (s, 1H, mal-NH), 6.27 (s, 1H, 9-H), 5.40 (d, 1H,J=6.8, NH), 4.67 (ddd, 1H, J=5.1, 5.4 and 6.8, H-4), 3.80 (s, 3H, H₃-6),3.48 (dd, 1H, J=5.1 and 13.8, HH-7), 3.62 (dd, 1H, J=5.4 and 14.1, HH-7)1.45 (s, 9H, 3×H₃-1); δ_(C) (125 MHz, CDCl₃) 170.2 (C═O), 168.9 (C═O),167.6 (C═O), 155.2 (C═O), 155.9 (C8), 119.7 (C9), 81.1 (C2), 53.3 (C6),52.7 (C4), 34.0 (C7), 28.3 (3×C1); IR (solid, cm⁻¹) 3236 (w), 1715 (s);MS (CI+) m/z, (relative intensity): 331 ([M+H], 5), 275 (20), 231 (100);Mass calcd for [C₁₃H₁₈O₆N₂S]+H requires 331.0964 Found 331.0968 (CI+);²⁰α_(D): −41.9° (c=1.0, Methanol); m.p. 145-147° C.; UV (Acetonitrile)ε₂₄₅=14200 and ε₃₃₉=8600 cm⁻¹M⁻¹d³.

Reference Example 40: Preparation of N-Boc-Cys(N′-Me-Mal)-OMe

To a stirring solution of N-Boc-Cys-OMe (32 mg, 0.136 mmol) in methanol(4 mL) was added sodium acetate (82 mg, 0.408 mmol). To this was addedN-methyl bromomaleimide (25.8 mg, 0.136 mmol) in methanol (4 mL) over 10minutes. The solvent was removed in vacuo purification by flashchromatography (gradient elution in 10% ethyl acetate in petroleum etherto 30% ethyl acetate in petroleum ether) to afford the desired compoundas a pale white powder (39.3 mg, 0.114 mmol) in 84% yield. δ_(H) (500MHz, CDCl₃) 6.26 (s, 1H, H-9), 5.36 (d, 1H, J=6.3, ‘Boc’ NH), 4.66 (m,1H, H-4), 3.79 (s, 3H, H₃-6), 3.46 (dd, 1H, J=5.0 and 5.2, HH-7), 3.35(dd, 1H, J=5.1 and 13.7, HH-7), 3.00 (s, 3H, H₃-13), 1.44 (s, 9H,3×H₃-1); δ_(C) (125 MHz, CDCl3) 170.2 (C═O), 169.5 (C═O), 167.9 (C═O),155.0 (C═O), 149.9 (C8), 118.7 (C9), 80.9 (C2), 53.1 (C6), 52.7 (C4),33.8 (C7), 28.3 (3×C1), 24.1 (C13); IR (solid, cm⁻¹) 3368 (m), 2977 (m),1695 (s); MS (ES+) m/z (relative intensity): 311 (M+, 100); Mass calcdfor C₁₄H₂₀N₂O₆NaS requires 367.0940. Found: 367.0931; ²⁰α_(D): −18.55o(c=1.0, Methanol); m.p. 101-103° C.

Example 9: Preparation of 2,3-Di(N-Boc-Cys-OMe)succinimide (Mix ofdiastereomers)

To a stirred solution of bromomaleimide (50 mg, 0.28 mmol) in aqueousbuffer (100M sodium phosphate, 150 mM NaCl, pH 8.0): DMF, 95:5 (9.25 mL)was added N-Boc-Cys-OMe (660 mg, 2.81 mmol) in DMF (0.25 mL). After 5minutes the aqueous reaction mixture was extracted with ethyl acetate(3×25 mL) and the combined organic layers washed with saturated lithiumchloride solution (aq) (5×25 mL), water (25 mL) and brine (25 mL), dried(MgSO₄), filtered and the solvent removed in vacuo. Purification bycolumn chromatography (10-40% ethyl acetate in petroleum ether) afforded2,3-Di(N-Boc-Cys-OMe)succinimide (mix of diastereomers) as a yellow waxyoil (150 mg, 0.27 mmol, 94% yield), an inseparable 1:1 mix of twosymmetrical diastereomers; δ_(H) (400 MHz, CDCl₃) 8.62 (s, 1H, maleimideNH from one symmetrical diastereomer), 8.66 (s, 1H, maleimide NH fromone symmetrical diastereomer), 5.62 (d, 2H, J=8.4, 2×‘Boc’ NH from onesymmetrical diastereomer), 5.51 (d, 2H, J=8.0, 2×‘Boc’ NH from onesymmetrical diastereomer), 4.72-4.58 (m, 4×H-4 from both diastereomers),3.80 (s, 6H, 2×H₃-6 from one symmetrical diastereomer), 3.79 (s, 6H,2×H₃-6 from one symmetrical diastereomer), 3.68 (s, 2H, 2×H-8 from onesymmetrical diastereomer), 3.64 (s, 2H, 2×11-8 from one symmetricaldiastereomer), 3.46 (dd, 2H, J=4.8 and 12.0 Hz, 2×HH-7* from onesymmetrical diastereomer), 3.37 (dd, 2H, J=6.0 and 14.4, 2×HH-7^(†) fromone symmetrical diastereomer), 3.21 (dd, 2H, J=4.8 and 14.0 Hz,2×HH-7^(†) from one symmetrical diastereomer), 3.11 (dd, 2H, J=6.4 and14.0 Hz, 2×HH-7* from one symmetrical diastereomer), 1.463 (s, 18H,6×H₃-1 from one symmetrical diastereomer), 14.460 (s, 18H, 6×H₃-1 fromone symmetrical diastereomer); *—signals shown as part of the same ABsystem by HMQC data^(†)—signals shown as part of the same AB system byHMQC data

δ_(C) (125 MHz, CDCl₃) 174.32 (2×C═O), 171.25 (2×C═O), 155.33 (2×C═O),80.61 (2×C2), 80.58 (2×C2), 53.51 (2×C4), 53.18 (2×C4), 52.91 (2×C6),52.90 (2×C6), 48.45 (2×C8), 47.89 (2×C8), 34.66 (2×C7), 34.59 (2×C7),28.37 (6×C1), 28.36 (6×C1) Several carbon signals are missing due tooverlap of the diastereomers; IR (thin film, neat) 3348, 2978, 1719cm⁻¹; MS (EI) m/z (relative intensity): 566 ([M+H], 20), 564 ([M−H],100); Exact mass calcd for [C₂H₃₅N₃O₁₀S₂]−H requires 564.1669. Found564.1686.

Reference Example 41: Preparation of N-Ac-Cys(Mal)-Benzylamine

To N-Ac-Cys-Benzylamine (1.00 g, 4.00 mmol) above) in methanol (42 mL),was added bromomaleimide (777 mg, 4.37 mmol) in methanol (42 mL)dropwise over 5 minutes. After 10 minutes, solvent removed in vacuo andresidue subjected to flash chromatography using 10% ethyl acetate inpetroleum ether afford the desired compound as an off-white solid (429mg, 1.2 mmol) in 100% yield, based on 69% recovery of thebromomaleimide. δ_(H) (5 (00) MHz, MeOD) 7.32-7.20 (m, 511, 5×Ar—H),6.45 (s, 1H, H-12), 4.71 (t, 1H, J=7.3, H-3), 4.38 (d, 2H, J=2.7, H₂-5),3.40 (dd, 1H, J=7.0 and 13.6, HH-10), 3.25 (dd, 1H, J=7.2 and 13.6,HH-10), 1.99 (s, 3H, H₃-1); δ_(C) (125 MHz, MeOD) 173.51 (C═O), 172.22(C═O), 171.44 (C═O), 170.51 (C═O), 151.58 (C11), 139.48 (C6), 129.54(2×Ar—H), 128.51 (2×Ar—H), 128.26 (C9), 121.01 (C12) 53.04 (C3), 44.25(C5), 33.72 (C10), 22.42 (C1); IR (film, cm⁻¹) 3187 (w), 1717 (s), 1646(s); MS (ES+) m/z (relative intensity): 370 ([M+Na], 20), 337 (50), 325(90), 309 (100); Exact Mass Calcd for [C₆H₁₇N₃O₄SN]+Na requires m/z370.0873. Found 370.0852 (ES+); UV (Acetonitrile) ε₂₁₃=19400, ε₂₄₇=4800and ε₃₃₇=2700 cm⁻¹M⁻¹d³; White solid decomposes at 180° C.

Reference Example 42: Preparation of N-Methyl Hexylsulfanylmaleimide

To N-methyl bromomaleimide (100 mg, 0.53 mmol) and sodium acetatetrihydrate (70 mg, 0.53 mmol) in methanol (15 mL) was added hexanethiol(74 μL, 0.58 mmol) in methanol (100 mL) dropwise over 1 hour withvigorous stirring. After 5 minutes solvent was removed in vacuo.Purification by column chromatography (gradient elution in 10% ethylacetate in petroleum ether to 30% ethyl acetate in petroleum ether)afforded the desired compound as a bright yellow solid (99 mg, 0.44mmol) in 83% yield. δ_(H) (600 MHz, CDCl₃) 6.03 (s, 1H, H-2), 3.01 (s,3H, H₃-5), 2.89 (t, 2H, J=7.6, 2H, H₂-11), 1.76-1.71 (m, 2H, H₂-10),1.46-1.41 (m, 2H, H₂-9), 1.33-1.27 (m, 4H, H₂-7 and CH₂-8), 0.89 (t, 3H,J=6.5, H₃-6); δ_(C) (125 MHz, CDCl₃) 171.47 (C═O), 169.94 (C═O), 151.84(C3), 117.27 (C2), 31.92 (C11), 31.31 (CH₂), 28.64 (CH₂), 27.75 (CH₂),24.10 (C5), 24.10 (C7), 14.09 (C6); IR (oil, cm⁻¹) 2727 (w), 1708 (s);MS (FAB+) m/z (relative intensity): 250 ([M+Na], 40), 228 (35), 199(30), 176 (100); Exact Mass Calcd for [C₁₁H₁₇NO₂S]+Na requires m/z250.0878. Found 250.0880 (FAB+).

Reference Example 43: Preparation of 2.3 Dihexylsulfanylsuccinimide andHexylsulfanylmaleimide

Method A

To bromomaleimide (300 mg, 1.69 mmol) and sodium acetate (138 mg, 1.69mmol) in methanol (60 mL) was added hexanethiol (356 μL, 2.50 mmol).After 5 minutes solvent removed in vacuo and purification by flashchromatography (10% ethyl acetate in petroleum ether) afforded 2,3dihexanethiosuccinimide as a bright yellow paste (13 mg, 0.04 mmol) in2% and hexylsulfanylmaleimide as a cream powder (alkene 310 mg, 1.46mmol) in 86% yield.

2,3 Dihexylsulfanylsuccinimide

δ_(H) (500 MHz, CDCl₃) 8.21 (s, 1H, NH), 3.49 (s, 2H, 2×H-7), 2.89-2.83(m, 2H, 2×HH-6), 2.79-2.83 (m, 2H, 2×HH-6), 1.71-1.57 (m, 4H, 2×CH₂),1.44-1.37 (m, 4H, 2×CH₂), 1.34-1.26 (m, 8H, 4×CH₂), 0.89 (t, 6H, J=6.8,2×H₃-1); δ_(C) (125 MHz, CDCl₃) 174.60 (2×C═O), 48.23 (2×C7), 32.34(2×CH₂), 31.26 (2×CH₂), 28.99 (2×CH₂) 28.46 (2×CH₂), 22.56 (2×CH₂),14.27 (2×C1); IR (solid, cm⁻¹) 3198 (m), 2928 (m), 1703 (s); No mass ionfound.

Hexylsulfanylmaleimide

δ_(H) (500 MHz, CDCl₃) 7.35 (s, 1H, NH), 6.04 (s, 1H, H-8), 2.91 (t, 2H,H₂-6), 1.78-1.72 (m, 2H, H₂-5), 1.48-1.42 (m, 2H, CH₂), 1.33-1.30 (m,4H, 2×CH₂), 0.90 (t, 3H, J=6.9, H₃-1); δ_(C) (125 MHz, CDCl₃) 169.06(C═O), 167.69 (C═O), 152.74 (C7), 118.24 (C8), 32.06 (C6), 31.26 (CH₂),28.58 (CH₂), 27.70 (CH₂), 22.52 (CH₂), 14.03 (C1); IR (solid, cm⁻¹) 3200(m), 2918 (m), 1703 (s); MS (ES−) m/z (relative intensity): 212 ([M−H],100); Exact Mass Calcd for [C₁₀H₁₅NO₂S]−H requires m/z 212.0745 Found212.0753 (ES−); m.p. 99-101° (C; UV (Acetonitrile) ε₂₄₇=12000 andε₃₄₇=9500 cm⁻¹M⁻¹d³.

Method B

To bromomaleimide (300 mg, 1.69 mmol) and sodium acetate (138 mg, 1.69mmol) in methanol (100 mL) was added hexanethiol (237 μL, 1.69 mmol).After 5 minutes solvent removed in vacuo and purification by flashchromatography (10% ethyl acetate in petroleum ether) affordedhexylsulfanylmaleimide as a cream powder (362 mg, 1.69 mmol) in 100%yield.

Reference Example 44: Preparation of N-MethylenecyclohexaneHexylsulfanylmaleimide

To N-methylenecyclohexane bromomaleimide (50 mg, 0.19 mmol) in methanol(50 mL), was added hexanethiol (52 μL, 0.37 mmol) and sodium acetate (50mg, 0.37 mmol) in methanol (50 mL) dropwise over 5 minutes. After 10minutes, solvent removed in vacuo and residue subjected to flashchromatography (petroleum ether) to afford the desired compound as anoff-white solid (29 mg, 0.09 mmol) in 84% yield. δ_(H) (600 MHz, CDCl₃)6.01 (s, 1H, H-3), 6.27 (s, 1H, 9-H), 3.42 (d, 1H, J=6.8, NH), 4.67(ddd, 1H, J=5.1, 5.4 and 6.8, H-4), 3.80 (s, 3H, H₃-6), 3.48 (dd, 1H,J=5.1 and 13.8, HH-7), 3.62 (dd, 1H, J=5.4 and 14.1, HH-7) 1.45 (s, 9H,3×H₃-1); δ_(C) (125 MHz, CDCl₃) 170.23 (C═O), 16844 (C═O), 151.49 (C2),117.08 (C3), 44.36 (C16), 37.00 (C15), 31.91 (2×CH₂), 31.32 (2×CH₂),30.73 (CH₂), 28.66 (CH₂), 27.78 (CH₂), 26.33 (CH₂), 25.73 (2×CH₂), 22.58(CH₂), 14.10 (C6); IR (solid, cm⁻¹) 2927 (m), 1700 (s); MS (ES+) m/z,(relative intensity): 310 ([M+H], 100), 180 (40); Mass calcd for[C₁₇H₂₇O₂NS]+H requires 310.1841. Found 310.1828 (ES+).

Reference Example 45: Preparation of 3-Mercaptopropylthiomaleimide and1,5-Dithio-8-aza-bicyclo[5.3.0]decan-7,9-dione

To bromomaleimide (30 mg, 0.17 mmol) and sodium acetate (14 mg, 0.17mmol) in methanol (6 mL) was added 1,3-propanedithiol (17 μl, 0.17mmol). After five minutes solvent was removed in vacuo and purificationby flash chromatography (10% ethyl acetate in petroleum ether) afforded3-mercaptopropylthiomaleimide and 1,5-dithio,8-aza-bicyclo[5,3,0]decan-7,9-dione as a pale yellow powder that was amix of two inseparable isomers 3-mercaptopropylthiomaleimide (7 mg, 0.03mmol) in 21% yield, 1,5-dithio, 8-aza-bicyclo[5,3,0]decan-7,9-dione (12mg, 0.06 mmol) in 34% yield. 6 (500 MHz, MeOD) 6.28 (s, 1H, H-5), 4.41(s, 3.2H, 2×HH-10), 3.15 (t, 2H, J=7.3, H₂-3), 2.82-2.77 (m, 3.2H, CH₂),2.35 (t, 3.2H, J=13.1, CH₂), 2.30-2.25 (m, 2H, CH₂), 2.20-2.13 (m, 2H,CH₂), 1.91-1.83 (m, 3.2H, CH₂); δ_(C) (125 MHz, MeOD) 177.79 (2×C11),172.33 (C═O), 170.56 (C═O), 152.37 (C4), 120.30 (C5), 54.52 (2×C10),34.94 (2×C9), 32.16 (CH₂), 31.10 (CH₂) 30.96 (CH₂), 27.49 (CH₂); IR(solid, cm⁻¹) 3246 (m), 1703 (s); MS (ES−) m/z (relative intensity): 202([M−H], 100); Exact Mass Calcd for [C₇H₉NO₂S₂]−H requires m/z 201.9996.Found 201.9996 (ES−).

Reference Example 46: Preparation of N-Phenyl Hexylsulfanylmaleimide

To hexanethiol (111 μL, 0.79 mmol) and sodium acetate trihydrate (108mg, 0.79 mmol) in methanol (60 mL) was added in N-phenylmonobromomaleimide (200 mg, 0.79 mmol) in methanol (60 mL) dropwise over1 hour with vigorous stirring. After 5 minutes solvent was removed invacuo. Purification by column chromatography (gradient elution in 10%ethyl acetate in petroleum ether to 30% ethyl acetate in petroleumether) afforded the desired compound as a pale yellow solid (109 mg,0.38 mmol) in 48% yield. δ_(H) (600 MHz, CDCl₃) 7.45 (dd, 2H, J=7.1 and8.0, 2×H-12), 7.36 (d, 2H, J=6.0, H-11), 7.35 (d, 2H, J=8.1, 2×H-13),6.19 (s, 1H, H-2), 2.96 (t, 2H, J=7.9, H₂-10), 1.81-1.76 (m, 2H, H₂-9),1.50-1.45 (m, 2H, H₂-8), 1.34-1.32 (m, 4H, H₂-6 and H₂-7), 0.91 (t, 3H,J=6.9, H₃-5); δ_(C) (125 MHz, CDCl₃) 168.59 (C═O), 166.96 (C═O), 152.20(C3), 131.53 (C14), 129.21 (2×Ar—H), 127.93 (C11), 126.09 (2×Ar—H),117.24 (C2), 32.03 (C10), 31.33 (CH₂), 28.68 (CH₂), 27.78 (Cl₂), 22.59(CH₂), 14.11 (C5); IR (oil, cm⁻¹) 2931 (w), 1703 (s); MS (CI+) m/z(relative intensity): 290 ([M+H], 100); Exact Mass Calcd for[C₁₆H₂₀NO₂S]+H requires m/z 290.1215. Found 290.1224 (CI+).

Reference Example 47: Preparation of Phenylthiomaleimide

To thiophenol (57 μL, 0.56 mmol) and sodium acetate trihydrate (136 mg,0.56 mmol) in methanol (30 mL) was added in monobromomaleimide (100 mg,0.56 mmol) in methanol (30 mL) dropwise over 1 hour with vigorousstirring. After 5 minutes solvent was removed in vacuo. Purification bycolumn chromatography (gradient elution in 10% ethyl acetate inpetroleum ether to 30% ethyl acetate in petroleum ether) afforded thedesired compound as a pale yellow solid (22 mg, 0.11 mmol) in 19% yield.δ_(H) (600 MHz, CDCl₃) 7.56 (dd, 2H, J=1.6 and 7.8, 2×H-7), 7.50-7.48(m, 3H, 3×Ar), 5.63 (s, 1H, H-2); δ_(C) (125 MHz, CDCl₃) 169.42 (C═O),167.98 (C═O), 153.60 (C3), 134.45 (2×Ar—H), 130.68 (C5), 130.42(2×Ar—H), 127.27 (C8), 119.91 (C2); IR (oil, cm⁻¹) 3265 (m), 1770 (m),1701 (s); MS (CI+) m/z (relative intensity): 206 ([M+H], 100), 111 (40);Exact Mass Calcd for [C₁₀H₇NO₂S]+H requires m/z 206.0276. Found 206.0273(CI+).

Reference Example 48: Preparation of1,4-Dithia-7-aza-spiro[4.4]nonane-6,8-dione

To bromomaleimide (30 mg, 0.17 mmol) and sodium acetate (14 mg, 0.17mmol) in methanol (6 mL) was added 1,2-ethanedithiol (17 μl, 0.17 mmol).After five minutes solvent removed in vacuo and purification by flashchromatography (10% ethyl acetate in petroleum ether) afforded thedesired compound as a pale yellow powder (13 mg, 0.07 mmol) in 41%yield. δ_(H) (500 MHz, CDCl₃) 8.39 (s, 1H, NH), 3.75-3.69 (m, 2H, HH-2and HH-3), 3.60-3.53 (m, 2H, HH-2 and HH-3), 3.30 (s, 2H, H₂-9); δ_(C)(125 MHz, CDCl₃) 177.93 (C═O), 172.76 (C═O), 61.23 (C5), 43.12 (C9),41.05 (C2 and C3); IR (solid, cm⁻¹) 3290 (m), 1703 (m), 1629 (s); MS(ES−) m/z (relative intensity): 188 ([M−H], 100); Exact Mass Calcd for[C₆H₇NO₂S₂]−H requires m/z 187.9840. Found 187.9839 (ES−); m.p. 112-115°C.

Example 10: Preparation of (S)-methyl2-(tert-butoxycarbonylamnino)-3-(1-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-ylthio)propanoate

Dansyl-bromomaleimide (100 mg) was dissolved in methanol (200 ml) bybriefly heating the stirring solution using a heat gun. To the resultingpale yellow solution, N-Boc-Cys-OMe (22 μl, 0.1 mmol, 0.5 eq) and sodiumacetate (14.5 mg, 0.1 mmol, 0.5 eq)) were added over 3 hours. Thereaction was monitored by TLC (eluent: 40% EtOAc: 60% Petroleum ether)Once the addition was complete the methanol was removed in vacuo toyield a yellow oil. Purification by column chromatography yielded thedesired product (48.29 mg, 0.08 mmol, 79.7%). ¹H NMR (600 MHz CDCl₃):δ_(H) 8.54 (d, J=8.56 Hz, 1H, CH), 8.21 (d, 1H, J=8.27 Hz, CH), 8.13 (d,1H, J=8.57 Hz, CH), 7.55 (m, 2H, 2×CH), 7.25 (d, J=7.60 Hz, 1H, CH),5.92 (s, 1H, (7H), 4.44 (m, 1H, HN—CH—CO), 3.77 (s, 3H, OMe), 3.48 (m,2H, CH₂), 3.44 (m, 2H, CH₂), 3.38 (s, 6H, 2×CH₃), 3.13 (t, J=5.79, 2H,S—CH₂) 2.88 (s, 9H, 3×CH₃). ¹³C NMR (600 MHz CDCl₃): 173.08, 170.52,169.69, 168.96 (4×C═O), 157.73 (—C═CH), 153.16, 150.71, 136.48, 131.39,131.25, 131.18, 130.74, 130.65, 129.31, 124.35, 120.61, 119.22, 81.12,53.58, 52.95, 45.89, 41.53, 33.98, 28.66. IR: 3324.7 cm⁻¹, 1775 cm⁻¹,[M+H]+: 605.1756, calculated; 605.1740

Example 11: Preparation of(2R,2′R)-dimethyl3,3′-(1-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethyl)-2,5-dioxopyrrolidine-3,4-diyl)bis(sulfanediyl)bis(2-(^(t)butoxycarbonylamino)propanoate)

Dansyl/maleimide/cysteine adduct (15 mg, 0.0247 mmol, 1 eq) wasdissolved in methanol (100 ml). To the resulting clear solution,N-Boc-Cys-OMe (3.1 μl, 0.0247 mmol, 1 eq) was added over 1 hour. Thereaction was monitored by TLC (eluent: 40% EtOAc:60% Petroleum ether)Once the addition was complete the methanol was removed in vacuo toyield the desired product (12.51 mg, 0.08 mmol, 60%). ¹H NMR (600 MHzCDCl3): δ_(H) 8.44 (d, J=8.56 Hz, 1H, CH), 8.13 (m, 2H, 2×CH), 7.49 (m,2H, 2×CH), 7.17 (m, 2H, 2×CH), 4.49 (bs, 1H, HN—CH—CO), 3.77 (s, 3H,OMe), 3.48 (m, 2H, CH₂), 3.44 (m, 2H, CH₂), 3.38 (s, 6H, 2×CH₃), 3.13(t, J=5.79, 2H, S—CH₂) 2.88 (s, 9H, 3×CH₃).

Example 12: Preparation of Di-Dansyl-Cystamine-Maleimide

A round bottomed flask was charged with di-dansyl cystamine (100 mg,0.16 mmol), TCEP (46 mg, 1 eq) and MeOH (10 ml). The reaction mixturewas stirred at ambient temperature under argon for 3 hrs.Dibromomaleimide (36 mg, 0.9 eq), in MeOH (5 ml) was then added to thereaction mixture. After 30 mins NaOAc (56 mg, 4 eq), was added to thereaction mixture and the solvent evaporated in vacuo. The residue wasworked up with DCM and brine. The organic layers were combined, dried(MgSO₄), filtered and concentrated in vacuo. Purification by flashchromatography (silica gel, 0-20% EtOAc-DCM) afforded the desiredcompound as a yellow gum (40 mg, 40%). ¹HNMR (CDCl₃ 600 MHz), δ8.5 (2H,d J 8.5 Hz aromatic H's), δ8.2 (4H, m aromatic H's), δ7.53 (1H, s CONH),δ7.46 (4H, m, aromatic H's), δ7.1 (2H, d, J 7.4 Hz aromatic H's), δ5.65(2H, t, J 6.27 SO₂NH), δ3.3 (4H, t, J 6.0 SCH₂), δ3.17 (4H, q, J 6.0NHCH₂), δ2.8 (12H, s NCH₃); ¹³CNMR (CDCl₃, 150 MHz), δ165.9, 152.0,136.5, 134.7, 130.7, 129.94, 129.85, 129.62, 129.57, 128.63, 123.3,118.8, 115.4, 45.5, 43.6, 31.8; IR (cm⁻¹) 3288 (br) 1720 (s) MS (Na+)m/z relative intensity: 736 (M, 100); Exact mass calculated for[C₃₂H₃₅N₅O₆NaS₄] requires m/z 736.1368. Found 736.1390 (Na+).

Reference Example 49: Preparation of Bromo-Dansyl-Cystamine-Maleimide

A round bottomed flask was charged with di-dansyl cystamine (48 mg, 0.08mmol), TCEP (23 mg, 1 eq), and MeOH (10 ml). The reaction mixture wasstirred at ambient temperature under argon for 3 hrs. Dibromomaleimide(41 mg, 2 eq) in MeOH (10 ml), was added to the reaction mixture. After16 hrs, the reaction mixture was concentrated in vacuo. The residue wasworked up with DCM and brine. The organic layers were combined, dried(MgSO₄) and purified by flash chromatography (silica gel, 0-15%EtOAC-DCM) to yield the desired compound (17 mg, 22%). ¹HNMR (CDCl₃, 600MHz), δ8.5 (1H, d J 8.5 Hz aromatic H's), δ8.2 (2H, m aromatic H's),δ7.6 (1H, s CONH), δ7.53 (2H, m, aromatic H's), δ7.15 (1H, d, J 7.4 Hzaromatic H's), δ5.30 (1H, t, J 5.6 SO₂NH), δ3.38 (2H, t, J 6.3 SCH₂),δ3.26 (2H, q, J 6.3 NHCH₂), δ2.88 (6H, s NCH₃); ¹³CNMR (CDCl₃ 150 MHz),δ165.5, 162.9, 152.2, 142.5, 134.5, 130.95, 129.94, 129.92, 129.5,128.7, 123.3, 119.0, 118.5, 115.4, 45.5, 43.7, 30.5; IR (cm⁻¹) 3295 (br)1726 (s) MS (ES+) m/z relative intensity: 485 (M, 100); Exact masscalculated for [C₁₈H₁₉N₃O₄S₂Br] requires m/z 484.0000. Found 783.9982.

Reference Example 50: Preparation of Dansyl-Cystamine-Maleimide

A round bottomed flask was charged with di-dansyl cystamine (100 mg,0.16 mmol), TCEP (46 mg, 1 eq), and MeOH (10 ml). The reaction mixturewas stirred at ambient temperature under argon for 3 hrs. Bromomaleimide(56 mg, 2 eq) in MeOH (5 ml), was added to the reaction mixture. After16 hrs, the reaction mixture was concentrated in vacuo. The residue wasworked up with DCM and brine. The organic layers were combined, dried(MgSO₄) and purified by flash chromatography (silica gel, 0-30%EtOAC—CHCl₃) to yield the desired compound (73 mg, 55%). ¹HNMR (CDCl₃,600 MHz), δ8.5 (1H, d J 8.5 Hz aromatic H's), δ8.2 (2H, m aromatic H's),δ8.1 (1H, s CONH), δ7.5 (2H, m, aromatic H's), δ7.15 (1H, d, J 7.5 Hzaromatic H's), δ6.0 (1H, s, CO₂CH) δ5.89 (1H, t, J 6.4 SO₂NH), δ3.20(2H, q, J 6.7 NHCH₂), δ3.99 (2H, t, J 6.9 SCH₂), δ2.86 (6H, s NCH₃);¹³CNMR (CDCl₃, 150 MHz), δ169.6, 168.0, 152.0, 150.7, 134.4, 131.0,129.9, 129.7, 129.5, 128.8, 123.4, 119.3, 118.7, 115.6, 45.5, 41.0,31.8; IR (cm⁻¹) 3277 (br) 1720 (s) MS (ES−) m/z relative intensity: 404(M, 100); Exact mass calculated for [C₁₈H₁₈N₃O₄S₂] requires m/z404.0739. Found 404.0733.

Example 13: Preparation ofN-Propionic-Acid-Methyl-Ester-Di-Dansyl-Cystamine-Maleimide

A round bottomed flask was charged with di-dansyl cystamine (132 mg,0.214 mmol), TCEP (61 mg, 1 eq) and MeOH (10 ml). The reaction mixturewas stirred at ambient temperature under argon for 3 hrs. Thedibromomaleimide (70 mg, 1 eq), in MeOH (5 ml) was then added to thereaction mixture. After 30 mins NaOAc (88 mg, 5 eq), was added to thereaction mixture and the solvent evaporated in vacuo. The residue wasworked up with DCM and brine. The organic layers were combined, dried(MgSO₄), filtered and concentrated in vacuo. Purification by flashchromatography (silica gel, 0-20% EtOAc-DCM) afforded the desiredcompound as a yellow gum (32 mg, 20%). ¹HNMR (CDCl₃, 300 MHz), δ8.5 (2H,d J 8.5 Hz aromatic H's), δ8.2 (4H, m aromatic H's), δ7.53 (1H, s CON),δ7.45 (4H, m, aromatic H's), δ7.1 (2H, d, J 7.5 Hz aromatic H's), δ5.7(2H, t, J 6.1 SO₂NH), δ3.75 (2H, t, J 7.0 CONCH₂), δ3.6 (3H, s, OCH₃),δ3.2 (4H, m, SCH₂), δ3.18 (4H, m, NHCH₂), δ2.9 (12H, s, NCH₃), δ2.6 (2H,t, J 7.1 NHCH₂); ¹³CNMR (CDCl₃, 75 MHz), δ171.3, 165.9, 135.8, 134.8,130.5, 129.8, 129.5, 129.4, 128.4, 123.3, 119.0, 115.3, 51.9, 45.5,43.5, 34.3, 32.6 31.9; IR (cm⁻¹) 3295 (br) 2948 (br) 1702 (s) MS (ES−)m/z relative intensity: 798 (M, 100); Exact mass calculated for[C₃₆H₄₀N₅O₈S₄] requires m/z 798.1760. Found 798.1715.

Reference Example 51: Preparation ofN-Propionic-Acid-Methyl-Ester-Bromo-Dansyl-Cystamine-Maleimide

A round bottomed flask was charged with di-dansyl cystamine (66 mg,0.107 mmol), TCEP (31 mg, 1 eq) and MeOH (10 ml). The reaction mixturewas stirred at ambient temperature under argon for 3 hrs. Thedibromomaleimide (70 mg, 0.5 eq), in MeOH (5 ml) was then added to thereaction mixture. After 16 hrs NaOAc (88 mg, 5 eq), was added to thereaction mixture and the solvent evaporated in vacuo. The residue wasworked up with DCM and brine. The organic layers were combined, dried(MgSO₄), filtered and concentrated in vacuo. Purification by flashchromatography (silica gel, 0-2% MeOH—CHCl₃) afforded the desiredcompound as a yellow gum (20 mg, 16%). ¹HNMR (CDCl₃, 300 MHz), δ8.5 (1H,m, aromatic H), δ8.2 (2H, m aromatic H's), δ7.5 (2H, m, aromatic H's),δ7.2 (1H, d, J 7.5 Hz aromatic H), δ5.2 (1H, t, J 6.1 SO₂NH), δ3.8 (2H,t, J 7.0 CONCH₂), δ3.7 (3H, s, OCH₃), δ3.4 (2H, m, SCH₂), δ3.3 (2H, m,NHCH₂), δ2.9 (6H, s, NCH₃), δ2.6 (2H, t, J 7.1 NHCH₂); ¹³CNMR (CDCl₃, 75MHz), δ170.95, 165.5, 163.3, 141.6, 134.5, 130.8, 129.8, 129.5, 128.5,123.2, 118.6, 115.3, 52.0, 45.4, 43.6, 34.8, 32.5 30.6; IR (cm⁻¹) 3296(br) 2948 (br) 1713 (s)

Example 14: Preparation ofN-Diethylene-Glvcol-Monomethyl-Ether-Di-Dansyl-Cystamine-Maleimide

A round bottomed flask was charged with di-dansyl cystamine (155 mg,0.25 mmol), TCEP (72 mg, 1 eq) and MeOH (10 ml). The reaction mixturewas stirred at ambient temperature under argon for 3 hrs.PEG-dibromomaleimide (100 mg, 1 eq), in MeOH (5 ml) was then added tothe reaction mixture. After 16 hrs NaOAc (102 mg, 5 eq), was added tothe reaction mixture and the solvent evaporated in vacuo. The residuewas worked up with DCM and brine. The organic layers were combined,dried (MgSO₄), filtered and concentrated in vacuo. Purification by flashchromatography (silica gel, 0-10% THF-DCM) afforded the desired compoundas a yellow gum (13 mg, 6%).

¹HNMR (MeOH, 300 MHz), δ8.5 (2H, m, aromatic H's), δ8.3 (2H, m aromaticH's), δ8.13 (2H, m, aromatic H's), δ7.5 (4H, m, aromatic H's), δ7.2 (2H,m, aromatic H's), δ3.5 (12H, m, CONCH₂. OCH₂), δ3.3 (3H, s, OCH₃), δ3.1(8H, m, SCH₂, NHCH₂), δ2.8 (12H, s, NCH₃); ¹³CNMR (CDCl₃, 150 MHz),δ167.4, 153.2, 136.9, 136.2, 131.3, 131.2, 130.9, 130.2, 129.6, 124.3,120.5, 116.4, 72.9, 71.4, 71.3, 71.1, 68.7, 59.1, 45.8, 44.5, 38.98,36.97; IR (cm⁻¹) 3323 (br) 2946 (br) 2946 (s) 1017 (s) MS (Na+) m/zrelative intensity: 882 (M, 100); Exact mass calculated for[C₃₉H₄₉N₅O₉NaS₄] requires m/z 882.2311. Found 882.2294 (Na+).

Reference Example 52: Preparation of Glu-Cys(Mal)-Gly

To glutathione (47 mg, 0.15 mmol) in methanol (3 mL) was addedbromomaleimide (30 mg, 0.15 mmol) in methanol (3 mL). After five minutessolvent removed in vacuo to afford the desired compound as a thickcolourless oil (62 mg, 0.15 mmol) in 100% yield. δ_(H) (500 MHz, MeOD)6.47 (s, 1H, H-12), 4.79 (dd, 1H, J=5.7 and 8.2, H-6), 4.06 (t, 1H,J=6.5, H-2), 3.95 (s, 2H, H₂-8), 3.49 (dd, 1H, J=5.8 and 13.9, HH-10),3.29 (dd, 1H, J=8.3 and 13.6, HH-10), 2.61 (t, 2H, J=7.1, H₂-4),2.29-2.15 (m, 2H, H₂-3); δ_(C) (125 MHz, MeOD) 174.68 (C═O), 172.81(C═O), 172.39 (C═O), 171.89 (C═O), 171.62 (C═O), 170.59 (C═O), 151.75(C11), 120.91 (C12), 53.79 (C6), 52.76 (C2), 42.01 (C8), 33.92 (C10)32.42 (C4), 27.03 (C3); IR (oil, cm⁻¹) 3259 (m), 2928 (m), 1717 (s); MS(ES−) m/z (relative intensity): 401 ([M−H], 100), 272 (30); Exact MassCalcd for [C₁₄H₁₈N₄O₈S]−H requires m/z 401.0767. Found 401.0773 (ES−);UV (Acetonitrile) ε₂₀₄=8100, ε₂₅₃=5600 and ε₃₄₂=1900 cm⁻¹M⁻¹d³.

Reference Example 53: Preparation of Preparation ofBoc-Cys(MeMal)-Phe-^(i)Pr

O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(344 mg, 0.82 mmol) was added to a stirred solution of(2R)-2-[(tert-butoxycarbonyl)amino]-3-[(1-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)sulfanyl]propanoicacid (313 mg, 0.95 mmol) and 1-hydroxybenzotriazole hydrate (139 mg) inDMF (2 mL) and the reaction was stirred at 21° C. for 3 mins. A solutionof (2S)-1-oxo-3-phenyl-1-(propan-2-ylamino)propan-2-ammoniumtrifluoroacetate (262 mg, 0.82 mmol) in DMF (1.5 mL) was added to thereaction mixture followed by N,N-diisopropylethylamine (294 μL, 1.64mmol) and the reaction stirred at 21° C. for 4 h. The solvent wasremoved in vacuo and the residue dissolved in EtOAc (60 mL) and washedwith 1 M HCl (×3), H₂O (×1), sat NaHCO₃ (×3), 10% LiCl (×1) and sat.NaCl (×1), dried (MgSO₄), filtered and the solvent removed in vacuo.Purification by precipitation (CHCl₃/petroleum ether 40-60) gave thedesired compound as a pale brown solid (359 mg, 0.69 mmol, 84% yield):¹H NMR (600 MHz, CD₃CN, 25° C.) δ 7.32-7.28 (m, 2H), 7.25-7.21 (m, 3H),7.11 (d, J=7.7 Hz, 1H), 6.46 (d, J=6.3 Hz, 1H), 6.42 (s, 1H), 4.46 (td,J=7.6, 6.5 Hz, 1H), 4.31 (td, J=7.3, 6.4 Hz), 3.88 (septets of doublet,J=6.6, 6.3 Hz, 1H), 3.30 (dd, J=13.7, 5.8 Hz, 1H), 3.16 (dd, J=13.7, 7.4Hz, 1H), 2.95 (dd, J=13.8, 7.5 Hz, 1H), 2.93 (s, 3H), 1.43 (s, 9H), 1.07(d, J=6.6 Hz, 3H), 1.02 (d, J=6.6 Hz, 3H); ¹³C NMR (151 MHz, CD₃CN, 25°C.) δ 169.39, 168.84, 168.77, 167.85, 155.17, 149.33, 136.77, 129.10,127.98, 126.30, 118.57, 79.49, 54.10, 52.62, 40.91, 37.44, 32.43, 27.15,22.95, 21.24, 21.17; IR (thin film) 3301, 2973, 1770, 1701, 1674, 1641,1525 cm⁻¹; LRMS (EI) 518 (24%, [M]⁺⁻), 432 (23), 219 (33), 149 (21) 110(27), 86 (37), 84 (100); HRMS (EI) calcd for C₂H₃₄N₄O₆S [M]⁺⁻ 518.2194,observed 518.2199.

Reference Example 54: Deprotection of Boc-Cys(MeMal)-Phe-^(i)Pr

Tris(2-carboxyethyl)phosphine hydrochloride (138 mg, 0.48 mmol) in 150mM phosphate buffer (pH 8, 25 mL) was added to a stirred solution ofBoc-Cys(MeMal)-Phe-^(i)Pr (50 mg, 97 μmol) in MeCN (25 mL) and thereaction stirred at 21° C. for 10 min. Synthesis of Boc-Cys-Phe-^(i)Prwas confirmed by LCMS (ES) 408.7 (100%).

Reference Example 55: Cloning and Expression of Grb2-SH2 L111C Mutant

Sequence of Grb2-SH2 L111C (residues 53-163): M G I E M K P H P W F F GK I P R A K A E E M L S K Q R H D G A F L I R E S E S A P G D F S L S VK F G N D V Q H F K V C R D G A G K Y F L W V V K F N S L N E L V D Y HR S T S V S R N Q Q I F L R D I E Q V P Q Q P T Y V Q A G S R S H H H HH H H Stop.

Calculated mass=14171

The DNA construct for the Grb2 SH2 domain contained the primary aminoacid sequence 53-163 and was cloned on plasmid QE-60 (Qiagen). The Grb2SH2 L111C mutant was constructed by site-directed mutagenesis(Stratagene Kit) using oligonucleotides coding for the mutated residue.Both constructs were expressed in Escherichia coli (M15[pREP4], Qiagen)using a T5 promoter and a C-terminal 6-His Tag was incorporated for thepurification. Cultures (1 L) were grown at 37° C. in T.B. from a singlecolony, and expression was induced with 1.0 mM IPTG when an O.D._(λ600)of 0.9 was reached. Cultures were allowed to express protein for roughly3 h before the cells were pelletised. Pellets were lysed in 0.1 M sodiumphosphate, 300 mM NaCl, 50 mM imidazole, pH 7.2 containing a proteaseinhibitor cocktail (Roche). The lysate was centrifuged, and thesupernatant was applied to a Ni-NTA column (Qiagen). Grb2-SH2 L111C waseluted from the Ni-NTA column with 0.1M sodium phosphate, 300 mM NaCl,200 mM imidazole at pH 7.2. The collected Grb2 SH2 L111C was ˜95% pureas visualized by Coomassie-stained SDS-PAGE. Dimerization of Grb2 SH2domain through domain-swapping has been previously observed. Dimeric andmonomeric Grb2-SH2 were separated on a Sephacryl S-100 column (320 mL)that had been pre-equilibrated with 0.1 M sodium phosphate and 150 mMNaCl at pH 8.0. Two peaks eluted, corresponding to the molecular weightsof monomer (˜14 kDa) and dimer (˜28 kDa) Grb2-SH2. Almost, 60% of theGrb2-SH2 L111C domain eluted from the column as monomer. Separatedmonomer and dimer were found to be surprisingly kinetically stable, asvery little interconversion was seen over a course of months at 4° C.The monomer was concentrated using Amicon® Ultra-4 centrifugal filterunits (Millipore) and the final concentration of the protein wasdetermined by absorbance at 280 nm using the extinction coefficientobtained by McNemar and coworkers (15,600M⁻¹). The protein was frozen at2 mg/mL concentration in 100 mL aliquots which were thawed as requiredfor experiments. The mass of the monomeric protein (mass 14170) wasobtained using ESI-MS.

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added Ellman'sreagent (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexedfor 1 s and maintained at 0° C. for 10 mins after which the mixture wasanalysed by LC-MS. Analysis showed that a single reaction had occurredyielding a single product with a mass of 14370 showing that C111 wasavailable for functionalisation.

Reference Example 56: Preparation of GrB2-SH2 DomainL111C/Bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide(5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 0° C. for 1 h. Analysis using LC-MS showed that thedesired product had been formed in quantitative conversion (mass 14266).

The mixture was treated with Ellman's reagent (5 μL, 282 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° (Cfor 10 mins after which the mixture was analysed by LC-MS. Analysisshowed that no reaction with Ellman's reagent was evident highlightingthat bromomaleimide functionalisation had occurred at C111.

Reference Example 57: Preparation of GrB2-SH2 DomainL111C/N-Methylbromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedN-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed that the desired product had been formed in quantitativeconversion (mass 14280).

The mixture was treated with Ellman's reagent (5 μL, 282 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 10 mins after which the mixture was analysed by LC-MS. Analysisshowed that no reaction with Ellman's reagent was evident highlightingthat N-methylbromomaleimide functionalisation had occurred at C111.

Reference Example 58: Phosphine-Mediated Reductive Cleavage of GrB2-SH2Domain L111C/Bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide(5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 0° C. for 1 h. Analysis using LC-MS showed a singleprotein species of mass 14265 which corresponded toprotein/bromomaleimide adduct.

The mixture was treated with TCEP.HCl (5 μL, 282 mM solution in H₂O) at0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 hafter which the mixture was analysed by LC-MS. Analysis showed that theprotein/bromomaleimide adduct had been cleanly cleaved yielding thedesired product (mass=14169) in 80% conversion

Reference Example 59: β-Mercaptoethanol-Mediated Reductive Cleavage ofGrB2-SH2 Domain L111C/Bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide(5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 0° C. for 1 h. Analysis using LC-MS showed a singleprotein species of mass 14265 which corresponded toprotein/bromomaleimide adduct.

The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution inH₂O), vortexed for 1 s and maintained at 37° C. for 4 h. Analysis showedthat the protein/bromomaleimide adduct had been cleanly cleaved yieldingthe desired product (mass=14173) in quantitative conversion.

Reference Example 60: Glutathione-Mediated Cleavage of GrB2-SH2 DomainL111C/Bromonaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide(5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 0° C. for 1 h. Analysis using LC-MS showed a singleprotein species of mass 14265 which corresponded toprotein/bromomaleimide adduct.

The mixture was treated with glutathione (5 μL, 282 mM solution in H₂O),vortexed for 1 s and maintained at 37° C. for 4 h. Analysis showed thatthe protein/bromomaleimide adduct had been cleanly cleaved yielding thedesired product (mass=14173) in quantitative conversion.

Reference Example 61: Phosphine-Mediated Reductive Cleavage of GrB2-SH2Domain L111C/N-Methylbromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedN-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed a single protein species of mass 14278 which corresponded toprotein/N-methylbromomaleimide adduct.

The mixture was treated with TCEP.HCl (5 μL, 282 mM solution in H₂O) at0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 hafter which the mixture was analysed by LC-MS. Analysis showed that theprotein/N-methylbromomaleimide adduct had been cleanly cleaved yieldingthe desired product (mass=14168) in 85% conversion.

Reference Example 62: β-Mercaptoethanol-Mediated Reductive Cleavage ofGrB2-SH2 Domain L111C/N-Methylbromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedN-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed a single protein species of mass 14280 which corresponded toprotein/N-methylbromomaleimide adduct.

The mixture was treated with 1-mercaptoethanol (5 μL, 282 mM solution inH₂O), vortexed for 1 s and maintained at 37° C. for 4 h. Analysis showedthat the protein/N-methylbromomaleimide adduct had been cleanly cleavedyielding the desired product (mass=14173) in quantitative conversion.

Reference Example 63: Glutathione-Mediated Cleavage of GrB2-SH2 DomainL111C/N-Methylbromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedN-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed a single protein species of mass 14280 which corresponded toprotein/N-methylbromomaleimide adduct.

The mixture was treated with glutathione (5 μL, 282 mM solution in H₂O),vortexed for 1 s and maintained at 37° C. for 4 h. Analysis showed thatthe protein/N-methylbromomaleimide adduct had been cleanly cleavedyielding the desired product (mass=14173) in quantitative conversion.

Reference Example 64: Ethanedithiol-Mediated Cleavage of GrB2-SH2 DomainL111C/bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide(5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 0° (C for 1 h. Analysis using LC-MS showed a singleprotein species of mass 14265 which corresponded toprotein/bromomaleimide adduct.

The mixture was treated with ethanedithiol (5 μL, 282 mM solution inH₂O), vortexed for 1 s and maintained at 37° C. for 4 h. Analysis showedthat the protein/bromomaleimide adduct had been cleanly cleaved yieldingthe desired product (mass=14173) in quantitative conversion.

Reference Example 65: Preparation of GrB2-SH2 DomainL111C/Bromomaleimide/2-Mercaptoethanol Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide(5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 0° C. for 1 h. Analysis using LC-MS showed a singleprotein species of mass 14265 which corresponded toprotein/bromomaleimide adduct.

The mixture was treated with 2-mercaptoethanol (5 μL, 2.82 mM solutionin H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0°C. for 3 h after which the mixture was analysed by LC-MS. Analysisshowed that the protein/bromomaleimide/2-mercaptoethanol adduct had beenformed (mass=14345) in 54% yield. The remaining material was GrB2-SH2domain L111C.

Reference Example 66: Preparation of GrB2-SH2 DomainL111C/N-Methylbromomaleimide/2-Mercaptoethanol Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedN-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed a single protein species of mass 14278 which corresponded toprotein/N-methylbromomaleimide adduct.

The mixture was treated with 2-mercaptoethanol (5 μL, 2.82 mM solutionin H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0°C. for 3 h after which the mixture was analysed by LC-MS. Analysisshowed that the desired product (mass=14359) had been formed in 90%yield. The remaining material was GrB2-SH2 domain L111C.

Example 15: Preparation of GrB2-SH2 DomainL111C/Bromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide(5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 0° C. for 1 h. Analysis using LC-MS showed a singleprotein species of mass 14265 which corresponds toprotein/bromomaleimide adduct.

The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3h after which the mixture was analysed by LC-MS. Analysis showed thatthe protein/bromomaleimide/glutathione adduct had been formed(mass=14574) in 44% conversion. The remaining material was GrB2-SH2domain L111C.

Example 16: Preparation of GrB2-SH2 DomainL111C/N-Methylbromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedN-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed a single protein species of mass 14278 which corresponded toprotein/N-methylbromomaleimide adduct.

The mixture was treated with 2-mercaptoethanol (5 μL, 2.82 mM solutionin H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0°C. for 3 h after which the mixture was analysed by LC-MS. Analysisshowed that the protein/N-methylbromomaleimide/glutathione adduct hadbeen formed (mass=14588) in 95% conversion. The remaining material wasGrB2-SH2 domain L111C.

Reference Example 67: Preparation of GrB2-SH2 DomainL111C/Dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed that the desired product had been formed in quantitative yield(mass 14345).

Reference Example 68: 2-Mercaptoethanol-mediated Reductive Cleavage ofthe GrB2-SH2 Domain L111C/Dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponded toprotein/dibromomaleimide adduct.

The mixture was treated with 2-mercaptoethanol (5 μL, 282 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 4 h after which the mixture was analysed by LC-MS. Analysis showedthat the protein/bromomaleimide adduct had been cleanly cleaved yieldingthe desired product (mass=14171) in quantitative yield.

Reference Example 69: Glutathione-Mediated Reductive Cleavage of theGrB2-SH2 Domain L111C/Dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponded toprotein/dibromomaleimide adduct.

The mixture was treated with glutathione (5 μL, 282 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4h after which the mixture was analysed by LC-MS. Analysis showed thatthe protein/bromomaleimide adduct had been cleanly cleaved yielding thedesired product (mass=14170) in quantitative yield.

Example 17: Preparation of GrB2-SH2 DomainL111C/Dibromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponded toprotein/dibromomaleimide adduct.

The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2h after which the mixture was analysed by LC-MS. Analysis showed thatthe desired product had been formed (mass=14573) in quantitativeconversion.

Example 18: Preparation of GrB2-SH2 DomainL111C/Dibromomaleimide/β-1-Thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponded toprotein/dibromomaleimide adduct.

The mixture was treated with β-1-thioglucose, sodium salt (5 μL, 2.82 mMsolution in H₂O) at 0° C. The mixture was vortexed for 1 s andmaintained at 0° C. for 2 h after which the mixture was analysed byLC-MS. Analysis showed that the desired product (mass=14461) was formedin near quantitative yield.

Example 19: Glutathione-Mediated Cleavage of GrB2-SH2 DomainL111C/Dibromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponded toprotein/dibromomaleimide adduct.

The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2h after which the mixture was analysed by LC-MS. Analysis showed thatthe protein/dibromomaleimide/glutathione adduct was the only proteinspecies present (mass=14573).

The mixture was treated with glutathione (5 μL, 282 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4h after which the mixture was analysed by LC-MS. Analysis showed thatthe desired product (mass=14173) was formed in quantitative yield.

Example 20: Glutathione-Mediated Cleavage of GrB2-SH2 DomainL111C/Dibromomaleimide/Glutathione Adduct at Physiologically RelevantGlutathione Concentration (5 mM)

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponded toprotein/dibromomaleimide adduct.

The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2h after which the mixture was analysed by LC-MS. Analysis showed thatthe protein/dibromomaleimide/glutathione adduct was the only proteinspecies present (mass=14573).

The mixture was treated with glutathione (5 μL, 100 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4h after which the mixture was analysed by LC-MS. Analysis showed thatthe desired product (mass=14173) was formed in quantitative yield.

Example 21: Glutathione-Mediated Cleavage of GrB2-SH2 DomainL111C/Dibromomaleimide/Glutathione Adduct at Physiologically RelevantGlutathione Concentration (1 mM)

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponded toprotein/dibromomaleimide adduct.

The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2h after which the mixture was analysed by LC-MS. Analysis showed thatthe protein/dibromomaleimide/glutathione adduct was the only proteinspecies present (mass=14573).

The solution of protein/dibromomaleimide/glutathione adduct wassubjected to a buffer swap (Micro Bio-Spin 6 Chromatography Column,Bio-Rad) yielding the adduct (95 μL, [adduct]0.2 mg/mL, 20 mM HEPES, 100mM KCl, 1 mM MgCl2, 1 mM EDTA, pH 7.4). To this was added glutathione (5μL, 20 mM solution in 20 mM HEPES, 100 mM KCl, 1 mM MgCl2, 1 mM EDTA, pH7.4). The mixture was vortexed for 1 s then maintained at 37° C. for 4h. Analysis showed that Grb2-SH2 (L111C) was formed (mass) 14170) inquantitative conversion.

Example 22: β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 DomainL111C/Dibromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponded toprotein/dibromomaleimide adduct.

The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2h after which the mixture was analysed by LC-MS. Analysis showed thatthe protein/dibromomaleimide/glutathione adduct was the only proteinspecies present (mass=14573).

The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 4 h after which the mixture was analysed by LC-MS. Analysis showedthat the desired product (mass=14172) was formed in quantitativeconversion.

Example 23: Glutathione-Mediated Cleavage of GrB2-SH2 DomainL111C/Dibromomaleimide/β-1-thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponded toprotein/dibromomaleimide adduct.

The mixture was treated with β-1-thioglucose (5 μL, 2.82 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 2 h after which the mixture was analysed by LC-MS. Analysis showedthat the protein/dibromomaleimide/β-1-thioglucose adduct was the onlyprotein species present (mass=14461).

The mixture was treated with glutathione (5 μL, 282 mM solution in H₂O)at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4h after which the mixture was analysed by LC-MS. Analysis showed thatdesired product was formed (mass=14173) in quantitative conversion.

Example 24: β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 DomainL111C/Dibromomaleimide/β-1-thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addeddibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MSshowed a single protein species of mass 14346 which corresponded toprotein/dibromomaleimide adduct.

The mixture was treated with β-1-thioglucose (5 μL, 2.82 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 2 h after which the mixture was analysed by LC-MS. Analysis showedthat the protein/dibromomaleimide/β-1-thioglucose adduct was the onlyprotein species present (mass=14461).

The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 4 h after which the mixture was analysed by LC-MS. Analysis showedthat desired product was formed (mass=14172) in quantitative conversion.

Reference Example 70: Preparation of GrB2-SH2 DomainL111C/N-Phenylbromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedN-phenylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed that the desired product had been formed in quantitative yield(mass 14351).

Reference Example 71: Preparation of GrB2-SH2 DomainL111C/N-Phenyldibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 20 mM MES,150 mM NaCl, pH 6) at 0° C. was added N-phenylbromomaleimide (5 μL, 2.82mM solution in DMF). The mixture was vortexed for 1 s then maintained at0° C. for 1 h. Analysis using LC-MS showed that the desired product hadbeen formed in quantitative yield (mass 14431).

Reference Example 72: β-Mercaptoethanol-mediated Cleavage of GrB2-SH2Domain L111C/N-Phenyldibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 20 mM MES,150 mM NaCl, pH 6) at 0° C. was added N-phenylbromomaleimide (5 μL, 2.82mM solution in DMF). The mixture was vortexed for 1 s then maintained at0° C. for 1 h. Analysis using LC-MS showed thatprotein/N-phenyldibromomaleimide adduct had been formed in quantitativeyield (mass 14431).

The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 4 h after which the mixture was analysed by LC-MS. Analysis showedthat desired product was formed (mass=14179) in quantitative conversion.

Example 25: Preparation of GrB2-SH2 DomainL111C/N-Phenyldibromomaleimide/β-1-Thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 20 mM MES,150 mM NaCl, pH 6) at 0° C. was added N-phenylbromomaleimide (5 μL, 2.82mM solution in DMF). The mixture was vortexed for 1 s then maintained at0° C. for 1 h. Analysis using LC-MS showed that theprotein/N-phenyldibromomaleimide adduct had been formed in quantitativeyield (mass 14431).

The mixture was treated with β-1-thioglucose (5 μL, 2.82 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 2 h after which the mixture was analysed by LC-MS. Analysis showedthat the protein/N-phenyldibromomaleimide/β-1-thioglucose adduct was theonly protein species present (mass=14547).

Example 26: β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 DomainL111C/N-Phenyldibromomaleimide/β-1-Thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 20 mM MES,150 mM NaCl, pH 6) at 0° C. was added N-phenylbromomaleimide (5 μL, 2.82mM solution in DMF). The mixture was vortexed for 1 s then maintained at0° C. for 1 h. Analysis using LC-MS showed that theprotein/N-phenyldibromomaleimide adduct had been formed in quantitativeyield (mass 14431).

The mixture was treated with β-1-thioglucose (5 μL, 2.82 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 2 h after which the mixture was analysed by LC-MS. Analysis showedthat the protein/N-phenyldibromomaleimide/β-1-thioglucose adduct was theonly protein species present (mass=14547).

The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 4 h after which the mixture was analysed by LC-MS. Analysis showedthat desired product was formed (mass=14178) in quantitative conversion.

Example 27: Preparation of GrB2-SH2 DomainL111C/Biotin-PEG-Bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedbiotin-PEG-bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixturewas vortexed for 1 s then maintained at 0° C. for 1 h. Analysis usingLC-MS showed that the desired product had been formed in quantitativeyield (mass 14634).

Example 28: β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 DomainL111C/Biotin-PEG-Bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedbiotin-PEG-bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixturewas vortexed for 1 s then maintained at 0° C. for 1 h. Analysis usingLC-MS showed that the protein/biotin-PEG-bromomaleimide adduct had beenformed in quantitative yield (mass 14634).

The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution inH₂O) at 37° C. The mixture was vortexed for 1 s and maintained at 37° C.for 4 h after which the mixture was analysed by LC-MS. Analysis showedthat desired product was formed (mass=14180) in quantitative conversion.

Example 29: Preparation of GrB2-SH2 DomainL111C/Biotin-PEG-Dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedbiotin-PEG-dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixturewas vortexed for 1 s then maintained at 0° C. for 2 h. Analysis usingLC-MS showed that the desired product had been formed in >80% yield(mass 14701).

Example 30: β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 DomainL111C/Biotin-PEG-Dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedbiotin-PEG-dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixturewas vortexed for 1 s then maintained at 0° C. for 1 h. Analysis usingLC-MS showed that the protein/biotin-PEG-dibromomaleimide adduct hadbeen formed in >80% conversion (mass 14701).

The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution inH₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C.for 4 h after which the mixture was analysed by LC-MS. Analysis showedthat desired product was formed (mass=14171) in >80% conversion.

Example 31: Pull-Down and Release of GrB2-SH2 DomainL111C/Biotin-PEG-Bromomaleimide Adduct onto Neutravidin Coated AgaroseBeads

To a solution of model protein (200 μL, [Protein] 1.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedbiotin-PEG-bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixturewas vortexed for 1 s then maintained at 0° C. for 1 h. Analysis usingLC-MS showed that the desired product had been formed in quantitativeyield (mass 14634).

Protein/biotin-PEG-bromomaleimide adduct (200 μL) and unmodified modelprotein (2 μL) were washed independently with PBS buffer (3×500 μL) in aconcentrator (Vivaspin, cut off 10 k) yielding protein solutions (300μL) (In). For each of the protein solutions obtained, neutravidin-coatedagarose beads (750 μL of 50% aqueous slurry) were washed with PBS (2×500μL). Protein solution (300 μL) was then added to the beads and themixture incubated at 4° C. for 30 mins. The mixture was centrifuged andthe flow through (FT) collected. The beads were washed with PBS (2×500μL) and both wash fractions collected (W1 and W2). Protein was releasedfrom the beads by incubation in PBS (300 μL) containingβ-mercaptoethanol (25 mM) for 2 h at 37° C. The sample was centrifugedand the eluant (EI) containing cleaved GrB2-SH2 domain L111C collected.The results are shown in FIG. 1.

The amount of protein recovered was determined as 44% by comparison witha protein series dilution via densitometry. However, correcting forirreversibly physisorbed protein (determined using the unmodifiedprotein control) the corrected recovery was 71%.

Example 32: Preparation of GrB2-SH2 Domain L111C/N-FluoresceinBromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 7.0) at 0° C. was added N-fluoresceinbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexedfor 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showedthat the desired product had been formed in 90% conversion (mass 14597).

Example 33: β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 DomainL111C/N-Fluorescein Bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 7.0) at 0° C. was added N-fluoresceinbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexedfor 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showedthat the protein/fluorescein bromomaleimide adduct had been formed in90% conversion (mass 14597).

The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution inH₂O) at 37° C. The mixture was vortexed for 1 s and maintained at 37° C.for 4 h after which the mixture was analysed by LC-MS. Analysis showedthat desired product was formed (mass=14171) in 87% conversion.

Example 34: Preparation of GrB2-SH2 Domain L111C/N-FluoresceinDibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-fluoresceindibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed that the desired product had been formed in 61% conversion (mass14675).

Example 35: β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 DomainL111C/N-Fluorescein Dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 7.0) at 0° C. was added N-fluoresceindibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture wasvortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MSshowed that the protein/fluorescein dibromomaleimide adduct had beenformed in 61% conversion (mass 14597).

The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution inH₂O) at 37° C. The mixture was vortexed for 1 s and maintained at 37° (7for 4 h after which the mixture was analysed by LC-MS. Analysis showedthat desired product was formed (mass=14171) in 85% conversion.

Reference Example 73: Preparation of GrB2-SH2 Domain L111C/BrDDPD Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added BrDDPD (5 μL,282 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 37° C. for 1 h. Analysis using LC-MS showed that thedesired product had been formed in quantitative yield (mass 14348).

Reference Example 74: β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2Domain L111C/BrDDPD Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added BrDDPD (5 μL,282 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 37° C. for 1 h. Analysis using LC-MS showed that theprotein/BrDDPD adduct had been formed in quantitative yield (mass14348).

The mixture was dialysed for 40 h at 4° C. (100 mM sodium phosphate, 150mM NaCl, pH 8.0) and treated with β-mercaptoethanol (5 μL, 2.82 Msolution in H₂O) at 37° C. The mixture was vortexed for 1 s andmaintained at 37° C. for 2 h after which the mixture was analysed byLC-MS. Analysis showed that desired product was formed (mass=14180) inquantitative conversion.

Reference Example 75: Preparation of GrB2-SH2 Domain L111C/DiBrDDPDAdduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added DiBrDDPD (5μL, 282 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 37° C. for 1 h. Analysis using LC-MS showed that thedesired product had been formed in quantitative yield (mass 14427).

Reference Example 76: β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2Domain L111C/DiBrDDPD Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added DiBrDDPD (5μL, 282 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 37° C. for 1 h. Analysis using LC-MS showed that theprotein/DiBrDDPD adduct had been formed in quantitative yield (mass14427).

The mixture was dialysed for 40 h at 4° C. (100 mM sodium phosphate, 150mM NaCl, pH 8.0) then treated with β-mercaptoethanol (5 μL, 2.82 Msolution in H₂O) at 37° C. The mixture was vortexed for 1 s andmaintained at 37° C. for 2 h after which the mixture was analysed byLC-MS. Analysis showed that desired product was formed (mass=14180) inquantitative conversion.

Example 36: Preparation of GrB2-SH2 DomainL111C/DiBrDDPD/β-1-Thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added DiBrDDPD (5μL, 282 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 37° C. for 1 h. Analysis using LC-MS showed that theprotein/DiBrDDPD adduct had been formed in quantitative yield (mass14427).

The mixture was dialysed for 40 h at 4° C. (100 mM sodium phosphate, 150mM NaCl, pH 8.0) then treated with β-1-thioglucose (5 μL, 28.2 mMsolution in H₂O) at 0° C. The mixture was vortexed for 1 s andmaintained at R₁ for 1 h after which the mixture was analysed by LC-MS.Analysis showed that the protein/DiBrDDPD/β-1-thioglucose adduct was theonly protein species present (mass=14543).

Example 37: β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 DomainL111C/DiBrDDPD/β-1-Thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added DiBrDDPD (5μL, 282 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 37° C. for 1 h. Analysis using LC-MS showed that theprotein/DiBrDDPD adduct had been formed in quantitative yield (mass14427).

The mixture was dialysed for 40 h at 4° C. (100 mM sodium phosphate, 150mM NaCl, pH 8.0) then treated with β-1-thioglucose (5 μL, 28.2 mMsolution in H₂O) at 0° C. The mixture was vortexed for 1 s andmaintained at RT for 1 h after which the mixture was analysed by LC-MS.Analysis showed that the protein/DiBrDDPD/β-1-thioglucose adduct was theonly protein species present (mass=14543).

The mixture was treated with β-mercaptoethanol (5 μL, 2.82 M solution inH₂O) at RT. The mixture was vortexed for 1 s and maintained at RT for 30mins after which the mixture was analysed by LC-MS. Analysis showed thatdesired product was formed (mass=14180) in quantitative conversion.

Example 38: Preparation of GrB2-SH2 Domain L111C/BrDDPD/β-1-ThioglucoseAdduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added BrDDPD (5 μL,282 mM solution in DMF). The mixture was vortexed for 1 s thenmaintained at 37° C. for 1 h. Analysis using LC-MS showed that theprotein/BrDDPD adduct had been formed in quantitative yield (mass14348).

The mixture was dialysed for 40 h at 4° C. (100 mM sodium phosphate, 150mM NaCl, pH 8.0) then treated with β-1-thioglucose (5 μL, 28.2 mMsolution in H₂O) at 0° C. The mixture was vortexed for 1 s andmaintained at 37° C. for 1 h after which the mixture was analysed byLC-MS. Analysis showed that the protein/BrDDPD/β-1-thioglucose adductwas formed in 17% conversion (mass=14543).

Reference Example 77: Preparation of GrB2-SH2 DomainL111C/Z-2,3-Dibromo-But-2-Enedioic Acid Dimethyl Ester Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedZ-2,3-dibromo-but-2-enedioic acid dimethyl ester (5 μL, 282 mM solutionin DMF). The mixture was vortexed for 1 s then maintained at 37° C. for1 h. Analysis using LC-MS showed that the desired product had beenformed (mass 14440).

Reference Example 78: β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2Domain L111C/Z-2,3-Dibromo-But-2-Enedioic Acid Dimethyl Ester Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mMsodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was addedZ-2,3-dibromo-but-2-enedioic acid dimethyl ester (5 μL, 282 mM solutionin DMF). The mixture was vortexed for 1 s then maintained at 37° C. for1 h. Analysis using LC-MS showed that the desired product had beenformed in quantitative yield (mass 14370).

The mixture was treated with β-mercaptoethanol (5 μL, 2.82 M solution inH₂O) at 37° C. The mixture was vortexed for 1 s and maintained at 37° C.for 2 h after which the mixture was analysed by LC-MS. Analysis showedthat desired product was formed (mass=14180) in quantitative conversion.

Example 39: Modification and Regeneration of Somatostatin

Preparation of Reduced Somatostatin

Lyophilised somatostatin (mass=1638) was solubilised in buffer (50 mMsodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) to yield a concentrationof 152.6 μM (0.25 mg/ml) and reduced with 1.1 equiv of TCEP for 1 h atambient temperature. Completeness of the reduction was confirmed byaddition of 4 equiv of dibromomaleimide to an aliquot of the sample andanalysis by LC-MS.

Bridging of Somatostatin with Halomaleimides and Derivatives

Reduced somatostatin was generated as described. 1.1 equiv of thehalomaleimides or dibromomaleimide derivates (100× stocks in 50 mMsodium phosphate, pH 6.2, 40% MeCN, 2.5-15.0% DMF) were added at ambienttemperature and the generation of product monitored over 1 h by LC-MS.The results are shown in FIG. 2.

Bridging of Somatostatin with Dithiomaleimides

Reduced somatostatin was generated as described. Various amounts ofdithiomaleimide (100× stocks in 50 mM sodium phosphate, pH 6.2, 40%MeCN, 2.5-7.5% DMF) were added at ambient temperature and the generationof product monitored over 1 h by LC-MS. The results are shown in FIG. 3.

Modification of Somatostatin with Bromomaleimide

Reduced somatostatin was generated as described. 2.1 equiv ofbromomaleimide (100× stock in 50 mM sodium phosphate, pH 6.2, 40% MeCN,7.5% DMF) were added at ambient temperature and complete conversion tothe di-addition product observed by LC-MS within 1 h.

Modification of Somatostatin with Dibromomaleic Anhydride

Reduced somatostatin was generated as described. 5 equiv ofdibromomaleic anhydride (in DMF) were added and the generation ofproducts monitored by LC-MS. 17.3% bridged somatostatin were generatedwithin 90 min.

Cleavage of Bridged Somatostatin with Various Reducing Agents

Maleimide bridged somatostatin was prepared as described. 100 equiv ofvarious reducing agents (1000× stock in 50 mM sodium phosphate, pH 6.2,40% MeCN, 2.5% DMF) were added and the generation of unmodified peptideand side products (mixed disulfides of the reducing agents with the freepeptide-cysteines) monitored at 4° C. over 2 d by LC-MS. Mixeddisulfides of somatostatin with GSH could only be detected by MALDI-TOFMS. The results are shown in FIG. 4.

Cleavage of Bridged Somatostatin with Various Amounts of DTT and2-Mercaptoethanol

Maleimide bridged somatostatin was prepared as described. Variousamounts of DTT or 2-mercaptoethanol (1000× stock in 50 mM sodiumphosphate, pH 6.2, 40% MeCN, 2.5% DMF) were added and the generation ofunmodified peptide and side products (mixed disulfides of the reducingagents with the free peptide-cysteines) monitored at 4° C. over 6 h byLC-MS. The results are shown in FIG. 5.

Catalysed Cleavage of Bridged Somatostatin

Maleimide bridged somatostatin was prepared as described. 20 equiv of2-mercaptoethanol (1000× stock in 50 mM sodium phosphate, pH 6.2, 40%MeCN, 2.5% DMF) were added followed by either buffer or 5 equiv ofsodium iodide or benzeneselenol (100× stock in 50 mM sodium phosphate,pH 6.2, 40% MeCN, 7.5% DMF) and the generation of unmodified peptide andside products (mixed disulfides of 2-mercaptoethanol or benzeneselenolwith the free peptide-cysteines) monitored at ambient temperature over20 min by LC-MS. The results are shown in FIG. 6.

Cleavage of N-Functionalised Maleimide Bridged Somatostatin

Somatostatin was reduced and bridged with N-functionalised maleimidederivates as described. 100 equiv of 2-mercaptoethanol (1000× stock in50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) were added and thegeneration of unmodified peptide and side products (mixed disulfides of2-mercaptoethanol with the free peptide-cysteines) monitored at 4° C.over 2 d by LC-MS. The results are shown in FIG. 7.

Cleavage of Di-Addition Product of Monobromomaleimide to Somatostatin

Reduced somatostatin was reacted with 2.1 equiv of monobromomaleimide togenerate the di-addition product. Next 100 equiv of 2-mercaptoethanol(1000× stock in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) wereadded and the generation of mono-addition product, unmodified peptideand side products (mixed disulfides of 2-mercaptoethanol with the freepeptide-cysteines) monitored at ambient temperature over 2.5 h by LC-MS.The results are shown in FIG. 8.

Comparable In Situ Bridging of Somatostatin

To somatostatin were added various amounts of dithiomaleimides (100×stock in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5-7.5% DMF) and thereaction was incubated at ambient temperature for 10 min. Next variousamounts of TCEP or benzeneselenol (100× stocks, freshly prepared in 50mM sodium phosphate, pH 6.2, 40% MeCN, 2.5-7.5% DMF) were added and thegeneration of bridged somatostatin was monitored over 1 h at ambienttemperature by LC-MS. The results are shown in FIG. 9.

In Situ PEGylation of Somatostatin

To somatostatin were added either 5 equiv ofN-PEG5000-dithiophenolmaleimide or 10 equiv ofN-PEG5000-dithiophenolmaleimide (100× stocks in 50 mM sodium phosphate,pH 6.2, 40% MeCN, 2.5% DMF) and the reaction was incubated at ambienttemperature for 10 min. Next 3 equiv of TCEP respectively 5 equiv ofbenzeneselenol (100× stocks, freshly prepared in 50 mM sodium phosphate,pH 6.2, 40% MeCN, 2.5-7.5% DMF) were added and the generation ofPEGylated somatostatin was monitored over 2 h at ambient temperature byLC-MS. The results are shown in FIG. 10.

Modification of Somatostatin with DiBrDDPD

Lyophilized somatostatin (mass=1638) was solubilized in buffer (50 mMsodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) to yield a concentrationof 152.6 μM (0.25 mg/mL) and reduced with 1.1 equiv of TCEP for 1 h at21° C. Completeness of the reduction was confirmed by LCMS (mass=1640).DiBrDDPD (100 mol eq) was added and the reaction maintained at 21° C.for 10 mins. Somatostatin/DiBrDDPD adduct was observed to formquantitative conversion (mass=1803).

Demonstration the Retained Biological Activity of Bridged SomatostatinsUsing Patch-Clamping

To examine whether the bridging modification had a deleterious effect onthe activity of the resultant somatostatin analogues we tested thedibromomaleimide bridged analogue, the PEGylated-dibromomaleimidebridged analogue, and the fluorescein dibromomaleimide-bridged analoguevia a patch clamp assay. HEK 293 cells expressing HKIR3.1/3.2 channeland human somatostatin receptor 2 were treated with these compounds, andwhole cell patch-clamp current recordings taken. All three analoguesinduced a robust activation of GIRK currents in an amplitude comparableto somatostatin itself. As a control when cells were treated withPertussis toxin, or by the GIRK inhibitor Tertiapin Q, currents werelargely inhibited. This data confirms that the bridged somatostatinanalogues retain the biological activity of somatostatin for agonism ofthe somatostatin receptor 2.

Cell Culture

Cell-culture methods and the generation of stable cell lines werecarried out as described in J Biol Chem 275, 921-9 (2000). HEK293 cells(human embryonic kidney cell line) stably expressing Kir3.1 and Kir3.2Achannels were maintained in minimum essential medium supplemented with10% foetal calf serum and 727 μg of G418 (Invitrogen), at 37° C. inhumidified atmosphere (95% O₂, 5% CO₂). Cells were transientlytransfected with SSTR2 DNA (Missouri S&T cDNA Resource Center) alongwith pEGFP-N1 (Clontech) for visualization of transfected cells usingepifluorescence. Transfections were performed with 5 μl of Fugene HD(Roche) and 800 ng SSTR2-DNA and 40 ng EGFP-DNA per 97 μl of cellculture medium (containing no serum or antibiotics).

Preparation of Somatostatin and Analogues for Patch-Clamp Experiments

Bridged somatostatins were prepared as described above. Somatostatin andits analogues were dialysed for 24 h at 4° C. in buffer (50 mM sodiumphosphate, pH 6.2) to remove the organic solvents. After dialysis theconcentration was determined and the peptides stored at 4° C. A finalconcentration of 20 μM somatostatin and analogues were used (dilutionwas done in the extracellular patch-clamp buffer).

Electrophysiology

Whole cell patch-clamp current recordings were performed with anAxopatch 200B amplifier (Axon Instruments) using fire-polished pipetteswith a resistance of 3-4 MΩ pulled from filamented borosilicated glasscapillaries (Harvard Apparatus, 1.5 mm OD×1.17 mm ID). Data was acquiredand analysed via a Digidata 1322A interface (Axon Instruments) andpCLAMP software (version 8.1, Axon Instruments). A fast perfusion systemwas used to apply somatostatin and analogues (Rapid Solution Changer,RSC-160, Bio-Logic France). Cells were clamped at −60 mV. Theextracellular solution was (mM): NaCl 80, KCl 60, CaCl₂ 2, MgCl₂ 1,HEPES 10, NaH₂PO₄ 0.33, glucose 10, pH 7.4; while the intracellularsolution was (mM): K gluconate 110, KCl 20, NaCl 10, MgCl₂ 1, MgATP 2,EGTA 2 GTP 0.3, pH 7.4. After agonist application, current activatedwith a delay “lag” followed by a rapid rise to peak amplitude “time topeak”. After removal of the agonist, the current decays back tobaseline. For each cell it was assessed if flow artifacts resulting fromthe pressure of drug application were present. This was done by applyingbath solution from one of the sewer pipes at the beginning of therecordings. Tertiapin, an inhibitor of GIRK current (Alomone), was usedat a final concentration of 100 nM. Cells were incubated overnight withpertussis toxin (Sigma, 100 ng/ml), an inhibitor of Gi/o proteins. Drugswere prepared as concentrated stocks solutions and kept at −20° C.

The results are shown in FIGS. 11 and 12.

Reference Example 79: Preparation of Propylaminomaleimide

To propylamine (75 μL, 1.09 mmol) and sodium acetate (92 mg, 1.12 mmol)in methanol (15 mL) was added bromomaleimide (200 mg, 1.12 mmol)dropwise in methanol (15 mL). After 10 minutes, solvent was removed invacuo and purification by flash chromatography (10% ethyl acetate inpetroleum ether) afforded the desired compound as a bright yellow waxysolid (82 mg, 0.53 mmol) in 49% yield. δ_(H) (500 MHz, CDCl₃) 7.36 (s,1H, NH), 5.45 (s, 1H, NH), 4.80 (d, 1H, J=1.3, H-5), 3.14 (dt, 2H, J=6.2and 7.2, H₂-3), 1.71-1.63 (m, 2H, H₂-2), 0.99 (t, 3H, J=7.4, H₃-1);δ_(C) (125 MHz, CDCl₃) 172.31 (C═O), 167.73 (C═O), 149.83 (C4), 85.29(C5), 46.16 (C3), 21.91 (C2), 11.42 (C1); IR (solid, cm⁻¹) 3190 (m),2962 (m), 1693 (m), 1627 (s); MS (EI) m/z (relative intensity): 154 (M+,60), 125 (98), 84 (100); Exact Mass Calcd for [C₇H₁₀N₂O₂]+ requires m/z154.0737. Found 154.0734 (EI); UV (Acetonitrile) ε₂₄₀=7400 and ε₃₄₈=5700cm⁻¹M⁻¹d³.

Reference Example 80: Preparation of But-3-enylaminomaleimide

To 3-butenylamine hydrochloride (200 mg, 1.12 mmol) and sodium acetate(184 mg, 2.24 mmol) in methanol (15 mL) was added bromomaleimide (200mg, 1.12 mmol) dropwise in methanol (15 mL). After 10 minutes, solventwas removed in vacuo and purification by flash chromatography (10% ethylacetate in petroleum ether) afforded the desired compound as a brightyellow waxy solid (142 mg, 0.85 mmol) in 76% yield. δ_(H) (500 MHz,CDCl₃) 7.10 (s, 1H, NH), 5.77 (tdd, 1H, J=6.9, 10.7 and 17.4, H-2), 5.38(s, 1H, NH), 5.18-5.15 (m, 2H, H₂-1), 4.83 (d, 1H, J=1.3, H-6), 3.24 (t,2H, J=6.7, H₂-4), 2.40 (dtd, 2H, J=1.2, 6.8 and 6.9, H₂-3); δ_(C) (125MHz, CDCl₃) 171.94 (C═O), 167.45 (C═O), 149.53 (C5), 133.89 (C2), 118.51(C1), 85.80 (C6), 43.30 (C4), 32.68 (C3); IR (solid, cm⁻¹) 3290 (m),1703 (m), 1629 (s); MS (ES−) m/z (relative intensity): 165 ([M−H], 100);Exact Mass Calcd for [C₈H₁₀N₂O₂]−H requires m/z 165.0659. Found 165.0664(ES−); m.p. 68-76° C.; UV (Acetonitrile) ε₂₄₁=8300 and ε₃₄₈=6100cm⁻¹M⁻¹d³.

Reference Example 81: Preparation of N-Methyl Propylaminomaleimide

To propylamine (52 μL, 0.78 mmol) and sodium acetate (64 mg, 0.78 mmol)in methanol (30 mL) was added N-methylmonobromomaleimide (150 mg, 0.78mmol) dropwise in methanol (30 mL). After 10 minutes, solvent wasremoved in vacuo and purification by flash chromatography (10% ethylacetate in petroleum ether) afforded the desired compound as a brightyellow waxy solid (41 mg, 0.24 mmol) in 31% yield. δ_(H) (500 MHz,CDCl₃) 5.43 (s, 1H, NH), 4.80 (s, 1H, H-2), 3.16-3.13 (m, 2H, H₂-9),2.98 (s, 3H, H₃-6), 1.71-1.64 (m, 2H, H₂-8), 0.99 (t, J=7.5, H₃-7);δ_(C) (125 MHz, CDCl₃) 172.71 (C═O), 167.66 (C═O), 149.51 (C3), 83.84(C2), 46.01 (C9), 23.44 (C6), 21.87 (C8), 11.38 (C7); IR (film, cm⁻¹)3317 (m), 2944 (w), 1698 (s), 1651 (s); MS (EI) m/z (relativeintensity): 168 (M+, 70), 139 (100), 111 (40); Exact Mass Calcd for[C₈H₁₂N₂O₂]+ requires m/z 168.0893. Found 168.0887 (EI); UV(Acetonitrile) ε₂₁₀=15900, ε₂₄₀=2800, ε₂₈₃=500 and ε₃₆₈=500 cm⁻¹M⁻¹d³.

Reference Example 82: Preparation of2,9-azatricyclo[5,3,0,0¹⁰⁻⁴]decan-1,3-dione

But-3-enylaminomaleimide (42 mg, 0.25 mmol) was dissolved inacetonitrile (25 mL), to provide a 0.01M solution. The resultingsolution was degassed for 30 minutes and irradiated in pyrex glasswarefor 4 minutes with stirring. Solvent was removed in vacuo andpurification by flash chromatography (gradient elution in ethyl acetateto 5% methanol in ethyl acetate) afforded the desired compound as anoff-white solid (39 mg, 0.23 mmol) in 93% yield. δ_(H)(500 MHz, CDCl₃)3.50 (ddd, 1H, J=2.6, 4.8 and 11.8, HH-8), 3.18-3.12 (m, 2H, HH-8 andH-6), 2.98 (dd, 1H, J=3.9 and 10.7, H-4), 2.21 (ddd, 1H, J=4.0, 8.6 and13.2, HH-5), 2.01 (ddd, 1H, 5.8, 10.5 and 13.4, HH-5), 1.79 (m, 2H,H₂-7); δ_(C) (125 MHz, CDCl₃) 179.04 (C═O), 178.95 (C═O), 70.85 (C10),48.43 (C8), 44.25 (C4), 43.82 (C6), 32.93 (C7) 24.96 (C5); IR (solid,cm⁻¹) 3198 (m), 2944 (m), 1701 (s); MS (EI) m/z (relative intensity):166 (M+, 45), 125 (100); Exact Mass Calcd for [C₈H₁₀N₂O₂]+ requires m/z166.07387. Found 166.07386 (EI); m.p. 110-113° C.

Reference Example 83: Preparation of (4SR, 6RS, 7SR)2-Aza-4-hexylsulfanyl-6-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione and(4RS, 5RS, 7RS)2-Aza-4-hexylsulfanyl-5-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile(22.5 mL) and acrylonitrile (2.5 mL) to provide a 0.005M solution. Theresulting solution was degassed for 30 minutes and irradiated in pyrexglassware for 5 minutes with stirring. Solvent was removed in vacuo andpurification by flash chromatography (gradient elution 10% ethyl acetatein petroleum ether to 50% ethyl acetate in petroleum ether) afforded(4SR, 6RS, 7SR)2-aza-4-hexylsulfanyl-6-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione as athick colourless oil (9 mg, 0.034 mmol) in 29% yield and (4RS, 7RS, 5RS)2-aza-4-hexylsulfanyl-5-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione as athick colourless oil (12 mg, 0.045 mmol) in 39% yield.

(4SR, 6RS, 7SR)2-Aza-4-hexylsulfanyl-6-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (500 MHz, CDCl₃) 3.53 (dt, 1H, J=1.4 and 8.1, H-6), 3.16-3.10 (m,2H, HH-5 and H-7), 2.89-2.80 (m, 2H, H₂-13), 2.56-2.50 (m, 1H, HH-5),1.67-1.55 (m, 4H, H₂-12 and H₂-11), 1.42-1.37 (m, 2H, H₂-10), 1.33-1.27(m, 2H, H₂-9), 0.89 (t, 3H, J=6.9, H₃-8); δ_(C) (125 MHz, CDCl₃) 174.49(C═O), 172.91 (C(═O), 116.82 (C4), 52.38 (C14), 44.16 (C6), 31.33 (C13),30.87 (C7), 30.29 (CH₂), 29.26 (CH₂), 28.64 (CH₂), 25.92 (CH₂), 22.82(C5), 14.11 (C8); IR (oil, cm⁻¹) 3223 (w), 2926 (w), 1778 (w), 1714 (s);MS (CI+) m/z (relative intensity): 267 ([M+H], 40), 213 (70), 180 (100);Exact Mass Calcd for [C₁₃1H₁₈N₂O₂S]+H requires m/z 267.1167. Found267.1175 (CI+).

(4RS, 7RS, 5RS)2-Aza-4-hexylsulfanyl-5-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (500 MHz, CDCl₃) 3.66 (dd, 1H, J=6.0 and 9.5, H-5), 3.23 (dd, 1H,J=5.2 and 10.9, H-7), 3.01-2.82 (m, 3H, HH-6 and H₂-13), 2.67 (ddd, 1H,J=5.3, 9.6 and 14.7, HH-6), 1.65-1.60 (m, 2H, H₂-12), 1.42-1.36 (m, 2H,H₂-11), 1.32-1.27 (m, 4H, H₂-9 and H₂-10), 0.88 (t, 3H, J=6.8. H₃-8);δ_(C) (125 MHz, CDCl₃) 175.08 (C═O), 174.82 (C═O), 117.13 (C4), 51.24(C14), 44.26 (C5), 31.36 (C13), 30.96 (C7), 29.82 (CH₂), 29.19 (CH₂),28.62 (CH₂), 25.72 (C6), 22.58 (CH₂), 14.26 (C8); IR (oil, cm⁻¹) 3247(w), 2927 (w), 1717 (s); MS (C1) m/z (relative intensity): 267 ([M+H],75), 214 (100), 180 (70); Exact Mass Calcd for [C₁₃H₁₈N₂O₂S]+H requiresm/z 267.1167. Found 267.1158 (CI).

Reference Example 84: Preparation of (5RS, 9SR)2-Aza-4-hexylsulfanyl-2-aza-tricylo[3.5.0.0^(5,9)]di-1,3-one

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile(22.5 mL) and cyclopentene (3 mL, 36 mmol) to provide a 0.005M solution.The resulting solution was degassed for 30 minutes and irradiated inpyrex glassware for 5 minutes with stirring. Solvent was removed invacuo and purification by flash chromatography (gradient elution 10%ethyl acetate in petroleum ether to 50% ethyl acetate in petroleumether) afforded the desired compound as a thick colourless oil (12 mg,0.045 mmol) in 77% yield, a 1:1 mix of two inseparable diastereomers.COSY analysis shows that certain signal arise from the same compound,denoted by subscripts ‘a’ and ‘b’, but the specific identity of eachdiastereomer is unknown. Overlap of signals prevents NOe analysis. δ_(H)(500 MHz, CDCl₃) 3.15-3.07 (m, 2H, H-5_(a) and H-10_(a)), 3.00 (t, 1H,J=6.8, H-5_(b)), 2.94 (td, 1H, J=3.9 and 6.6, H-9_(b)), 2.87-2.82 (m,2H, H-9_(a) and HH-16_(a)), 2.64-2.59 (m, 1H, HH-16_(b)), 2.52-2.47 (m,3H, H-10_(b), HH-16_(a) and HH-16_(b)), 2.07 (dd, 2H, J=6.3 and 6.9,HH-15_(a) and HH-15_(b)), 1.96-1.89 (m, 2H, HH-15_(a) and HH-15_(b))1.88-1.82 (m, 4H, H₂-7_(a) and H₂-7_(b)) 1.64-1.50 (m, 8H, H₂-6_(a),H₂-6_(b), H₂-8_(a) and H₂-8_(b)), 1.38-1.25 (m, 12H_(a), H₂-12_(a),H₂-12_(b), H₂-13_(a), H₂-13_(b), H₂-14_(a) and H₂-14_(b)), 0.89-0.86 (m,6H, H₃-11_(a) and H₃-11_(b)); δ_(C) (125 MHz, CDCl₃) 179.09 (C═O),177.12 (C═O), 176.93 (C═O), 171.83 (C═O), 51.31 (C4), 51.33 (C4), 50.68(C10), 45.32 (C10), 43.26 (C9), 41.70 (C5), 32.38 (CH₂), 30.98 (CH₂),30.95 (CH₂), 30.78 (CH₂), 28.92 (CH₂), 28.50 (CH₂), 28.30 (CH₂), 28.22(CH₂), 28.10 (CH₂), 28.13 (CH₂), 25.09 (CH₂), 22.14 (CH₂), 22.11 (CH₂),13.66 (2×C11) Several carbon signals are missing due to overlap of thediastereomers; IR (oil, cm⁻¹) 3120 (w), 2927 (m), 1711 (s), 1627 (s); MS(ES−) m/z (relative intensity): 280 ([M−H], 50), 212 (100); Exact MassCalcd for [C₁₅H₂₃NO₂S]−H requires m/z 280.1371. Found 280.1382 (ES−).

Reference Example 85: Preparation of4-Hexylsulfanyl-1-phenyl-1,7-dihydro-2H-azepine-3,6-dione and (4RS, 5SR,7RS) 2-Aza-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile(25 mL) to provide a 0.005M solution. The resulting solution wasdegassed for 30 minutes, styrene (133 μL, 1.2 mmol) added and thesolution irradiated in pyrex glassware for 5 minutes with stirring.Solvent was removed in vacuo and purification by flash chromatography(gradient elution in petroleum ether to 30% ethyl acetate in petroleumether) afforded4-hexylsulfanyl-1-phenyl-1,7-dihydro-2H-azepine-3,6-dione as a thickcolourless oil (11 mg, 0.034 mmol) in 30% yield and (4RS, 5SR, 7RS)2-aza-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione as a thickcolourless oil (26 mg, 0.082 mmol) in 70% yield

4-Hexylsulfanyl-1-phenyl-1,7-dihydro-2H-azepine-3,6-dione

δ_(H) (600 MHz, CDCl₃) 7.34-7.24 (m, 5H, 5×Ar—H), 6.15 (d, 1H, J=1.5,H-5), 4.11 (t, 1H, J=7.7, H-1), 3.01 (ddd, 1H, J=1.5, 7.8 and 15.8,HH-7), 2.96 (dd, 1H, J=7.8 and 15.6, HH-7), 2.36-2.26 (m, 2H, H₂-13),1.50-1.41 (m, 2H, H₂-12), 1.35-1.33 (m, 6H, H₂-9, H₂-10 and H₂-11), 0.85(t, 3H, J=7.0, H₃-8); δ_(C) (150 MHz, CDCl₃) 170.96 (C═O), 169.95 (C═O),147.26 (C4), 141.10 (C14), 129.41 (C5), 128.89 (2×Ar—H), 127.86 (C17),127.71 (2×Ar—H), 47.24 (C1), 32.47 (C13), 31.43 (CH₂), 29.19 (CH₂),28.61 (CH₂), 22.60 (CH₂), 14.10 (C8); IR (oil, cm⁻¹) 3288 (w), 2928 (w),1775 (w), 1717 (s); MS (FAB+) m/z (relative intensity): 340 ([M+Na],20), 329 (35), 207 (20), 176 (100); Exact Mass Calcd for [C₁₆H₂₃NO₂S]+Narequires m/z 340.1347. Found 340.1351 (FAB+).

(4RS, 5SR, 7RS)2-Aza-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (600 MHz, CDCl₃) 8.77 (s, 1H, NH), 7.39-7.31 (m, 5H, 5×Ar—H), 4.05(t, 1H, J=8.8, H-5), 3.17 (dd, 1H, J=3.4 and 10.9, H-7), 3.04 (ddd, 1H,J=8.4, 11.1 and 12.7, HH-6), 2.63 (ddd, 1H, J=3.6, 9.0 and 12.8, HH-6),2.43 (ddd, 1H, J=6.7, 7.9 and 11.3, HH-17), 2.13 (ddd, 1H, J=6.6, 8.0and 11.3, HH-17), 1.30-1.08 (m, 8H, H₂-13, H₂-14 and H₂-15 and H₂-16),0.83 (t, 3H, J=7.3, H₃-12); δ_(C) (150 MHz, CDCl₃) 178.76 (C═O), 177.62(C═O), 136.51 (C11), 128.77 (2×Ar—H), 128.70 (2×Ar—H), 128.03 (C8),57.17 (C4), 45.70 (C5), 43.87 (C7), 31.26 (C17), 28.70 (CH₂), 28.65(CH₂), 28.52 (CH₂), 26.22 (C6), 22.46 (CH₂), 14.10 (C12); IR (oil, cm⁻¹)3218 (w), 2926 (w) 1771 (m), 1703 (s); MS (FAB+) m/z (relativeintensity): 340 ([M+Na], 20), 199 (25), 176 (100); Exact Mass Calcd for[C₁₆H₂₃NO₂S]+Na requires m/z 340.1347 Found 340.1357 (FAB+).

Reference Example 86: Preparation of (4RS, 7SR, 5RS)2-Aza-4-hexylsulfanyl-5-carboxylic Acid methylester-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile(21.9 mL) and methyl acrylate (3.1 mL, 36 mmol) to provide a 0.005Msolution. The resulting solution was degassed for 30 minutes andirradiated in pyrex glassware for 5 minutes with stirring. Solvent wasremoved in vacuo and purification by flash chromatography (gradientelution in 10% ethyl acetate in petroleum ether to 50% ethyl acetate inpetroleum ether) afforded the desired compound as a thick colourless oil(17 mg, 0.056 mmol) in 48% yield. δ_(H) (600 MHz, CDCl₃) 8.50 (s, 1H,NH), 3.81 (s, 3H, H₃-8), 3.57 (dd, 1H, J=5.8 and 8.5, H-5), 3.18 (dd,1H, J=5.0 and 10.7, H-7), 3.11 (ddd, 1H, J=5.5, 11.0 and 12.9, HH-6),2.73 (dt, 1H, J=7.5 and 11.5, HH-15), 2.64 (dt, 1H, J=7.5 and 11.5,HH-15), 2.29 (ddd, 1H, J=5.2, 8.5 and 13.2. HH-6), 1.52-1.47 (m, 2H,H₂-14), 1.35-1.30 (m, 2H, H₂-13), 1.29-1.21 (m, 4H, H₂-11 and H₂-12),0.87 (t, 3H, J=6.7, H₃-10); δ_(C) (150 MHz, CDCl₃) 176.65 (C═O), 171.13(C═O), 170.48 (C═O), 52.59 (C8), 52.39 (C4), 44.56 (C7), 44.06 (C5),31.41 (C15), 29.73 (CH₂), 29.16 (CH₂), 28.68 (CH₂), 23.57 (C12), 22.57(C11), 14.11 (C10); IR (oil, cm⁻¹) 3244 (w), 2928 (w) 1778 (w), 1714(s); MS (FAB+) m/z (relative intensity): 322 ([M+Na], 100), 300 (30),214 (25); Exact Mass Calcd for [C₁₄H₂₁NO₄SN]+Na requires m/z 322.1089.Found 322.1082 (FAB+).

Reference Example 87: Preparation of (4RS, 5SR, 7RS)2-Aza-4-hexylsulfanyl-5-carboxylic Acid phenylester-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile(25 mL) to provide a 0.005M solution. The resulting solution wasdegassed for 30 minutes, phenyl acrylate (160 μL, 1.20 mmol) added andirradiated in pyrex glassware for 5 minutes with stirring. Solvent wasremoved in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded the desired compound as a thick colourless oil (21 mg, 0.058mmol) in 48% yield and hexylsulfanylmaleimide dimer (12 mg, 0.028 mmol)in 47% yield. δ_(H)(600 MHz, CDCl₃) 8.41 (s, 1H, NH), 7.41-7.39 (m, 2H,2×Ar-H), 7.27-7.25 (m, 1H, H-8), 7.20 (d, 1H, J=7.8, 2×Ar—H), 3.80 (dd,1H, J=5.1 and 8.5, H-5), 3.29 (dd, 1H, J=5.1 and 10.7, H-7), 3.20 (ddd,1H, J=5.5, 10.9 and 13.0, HH-6), 2.62 (dt, 1H, J=7.5 and 11.5, HH-18),2.73 (dt, 1H, J=7.5 and 11.5, HH-18), 2.40 (ddd, 1H, J=5.6, 8.8 and13.5, HH-6), 1.54-1.48 (m, 2H, H₂-17), 1.33-1.16 (m, 6H, H₂-14, Hz-15and H₂-16), 0.84 (t, 3H, J=6.9, H₃-13); δ_(C) (150 MHz, CDCl₃) 176.35(C═O), 176.19 (C═O), 168.85 (C═O), 150.66 (C11), 129.64 (2×Ar—H), 126.34(C8), 121.54 (2×Ar—H), 52.59 (C4), 44.78 (C7), 44.17 (C5), 31.36 (CH₂),29.94 (C18), 29.09 (CH₂), 28.68 (CH₂), 23.83 (C6), 22.56 (CH₂), 14.08(C13); IR (oil, cm⁻¹) 3213 (w), 2927 (w) 1757 (m), 1715 (s); MS (CI+)m/z (relative intensity): 362 ([M+H], 35), 268 (100), 149 (25); ExactMass Calcd for [C₁₉H₂₃NO₄S]+H requires m/z 362.1426. Found 362.1431(CI+).

Reference Example 88: Preparation of (4RS, 5SR, 7RS)2-Aza-4-hexylsulfanyl-5-(p-amino)phenyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile(25 ml) to provide a 0.005M solution. The resulting solution wasdegassed for 30 minutes, 4-vinyl aniline (136 μL, 1.2 mmol) added andirradiated in pyrex glassware for 5 minutes with stirring. Solvent wasremoved in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded the desired compound as a thick colourless oil (7 mg, 0.021mmol) in 17% yield. δ_(H) (600 MHz, CDCl₃) 8.17 (s, 1H, NH), 7.10 (d,2H, J=8.5, 2×Ar—H), 6.67 (d, 2H, J=8.5, 2×Ar—H), 3.94 (t, 1H, J=9.0,H-5), 3.13 (dd, 1H, J=3.7 and 11.1, H-7), 2.98 (ddd, 1H, J=8.8, 11.2 and12.9, HH-6), 2.58 (ddd, 1H, J=3.5, 9.1 and 12.8, HH-6), 2.42 (dt, 1H,J=7.5 and 11.5, HH-17), 2.17 (dt, 1H, J=7.5 and 11.5, HH-17), 1.34-1.29(m, 2H, H₂-16), 1.25-1.11 (m, 6H, H₂-13, H₂-14 and H₂-15), 0.80 (t, 3H,J=7.4, H₃-12); δ_(C) (150 MHz, CDCl₃) 178.64 (C═O), 177.48 (C═O), 146.28(C8), 129.81 (2×Ar—H), 126.20 (C11), 114.81 (2×Ar—H), 57.97 (C4), 45.48(C5), 43.79 (C7), 31.35 (CH₂), 29.07 (CH₂), 28.65 (C17), 26.41 (C6),22.54 (CH₂), 14.12 (C12); IR (oil, cm⁻¹) 3214 (w), 2928 (w) 1769 (m),1715 (s); MS (CI+) m/z (relative intensity): 333 ([M+H], 55), 119 (100);Exact Mass Calcd for [C₁₈H₂₄N₂O₂S]+H requires m/z 333.1637. Found333.1642 (CI+).

Reference Example 89: Preparation of4-Hexylsulfanyl-1-(m-nitro)phenyl-1,7-dihydro-2H-azepine-3,6-dione,(4RS, 5SR, 7RS)2-Aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dioneand (4RS, 5RS, 7RS)2-Aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile(25 mL) to provide a 0.005M solution. The resulting solution wasdegassed for 30 minutes, 3-nitrostyrene (136 μL, 1.2 mmol) added andirradiated in pyrex glassware for 5 minutes with stirring. Solvent wasremoved in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded4-hexylsulfanyl-1-(m-nitro)phenyl-1,7-dihydro-2H-azepine-3,6-dione as athick colourless oil (23 mg, 0.063 mmol) in 55% yield, (4RS, 5SR, 7RS)2-aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dioneas a thick colourless oil (0.5 mg, 0.001 mmol) in 1% yield (alongside4-hexylsulfanyl-1-(m-nitro)phenyl-1,7-dihydro-2H-azepine-3,6-dione), and(4RS, 5RS, 7RS)2-aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dioneas a thick colourless oil (12 mg, 0.33 mmol) in 21% yield (alongsidedimer).

2-Aza-4-hexylsulfanyl-1-(m-nitro)phenyl-1,7-dihydro-2H-azepine-3,6-dione

δ_(H) (600 MHz, CDCl₃) 8.22 (s, 1H, H-16), 8.14 (d, 1H, J=8.5, Ar—H),7.68 (t, 1H, J=7.6, Ar—H), 7.53 (t, 1H, J=7.8, H-19), 7.20 (s, 1H, NH),6.29 (s, 1H, H-5), 4.25 (t, 1H, J=7.9, H-1), 3.03 (dd, 1H, J=8.4 and15.4, HH-7), 2.98 (dd, 1H, J=7.4 and 15.2, HH-7), 2.37-2.26 (m, 2H,H₂-13), 1.30-1.24 (m, 2H, H₂-12), 1.21-1.08 (m, 6H, 3×CH₂), 0.81 (t, 3H,J=7.1, H₃-8); δ_(C) (150 MHz, CDCl₃) 170.70 (C═O), 169.54 (C═O), 148.60(C16), 146.35 (C4), 143.94 (C14), 133.83 (Ar—H), 129.96 (Ar—H), 129.86(C5), 122.96 (Ar—H), 122.59 (Ar—H), 46.78 (C1), 32.52 (C7), 31.56 (CH₂),31.37 (C13), 29.03 (CH₂), 28.52 (CH₂), 22.57 (CH₂), 14.11 (C8); IR (oil,cm⁻¹) 3282 (w), 2928 (m) 1775 (w), 1717 (s); Mass ion not found.

(4RS, 5SR, 7RS)2-Aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dionesignals are bold

δ_(H) (600 MHz, CDCl₃) 8.59 (s, 0.2H, NH), 8.22-8.16 (m, 0.4H, 2×Ar—H),8.15 (d, 1H, J=8.4, H-10), 8.07 (s, 1H, H-8), 7.84 (s, 1H, NH), 7.66 (d,0.2H, J=7.5, Ar—H), 7.62 (d, 1H, J=7.6, H-12), 7.56 (t, 0.2H, J=8.0,H-11), 7.53 (t, 1H, J=8.0, H-11), 4.14 (t, 0.2H, J=8.6, H-5), 4.10 (t,1H, J=9.4, H-5), 3.30 (dd, 1H, J=6.1 and 10.4, H-7), 3.23 (dd, 0.2H,J=3.0 and 11.6, H-7), 3.17 (dt, 1H, J=10.3 and 13.3, HH-6), 3.05 (ddd,0.2H, J=8.5, 11.1 and 13.1, HH-6), 2.73 (ddd, 0.2H, J=3.6, 9.0 and 12.9,HH-6), 2.68-2.56 (m, 3H, HH-6 and H₂-19), 2.45 (ddd, 0.2H, J=6.7, 8.1and 11.4, HH-19), 2.13 (ddd, 0.2H, J=6.8, 8.0 and 11.3, HH-19),1.40-1.35 (m, 2.4H, H₂-18 and H₂-18), 131-1.23 (m, 6H, H₂-15, H₂-16 andH₂-17), 1.21-1.08 (m, 1.2H, H₂-15, H₂-16 and H₂-17), 0.87 (t, 3H, J=6.9,H-14), 0.81 (t, 0.6H, J=7.1, H₃-14); δ_(C) (150 MHz, CDCl₃) 176.25(C═O), 174.16 (C═O), 148.44 (C9), 139.02 (C13), 133.88 (Ar—H), 129.69(Ar—H), 123.07 (Ar—H), 121.92 (Ar—H), 57.34 (C4), 46.76 (C5), 44.18(C7), 31.36 (CH₂), 30.21 (CH₂), 29.33 (C19), 28.68 (CH₂), 26.07 (C6),22.57 (CH₂) 14.11 (C14); IR (oil, cm⁻¹) 2934 (w), 1719 (s); MS (CI+) m/z(relative intensity): 363 ([M+H], 65), 214 (90), 180 (100); Exact MassCalcd for [C₁₈H₂₂N₂O₄S]+H requires m/z 363.1379. Found 363.1397 (CI+).

(4RS, 5RS, 7RS)2-Aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (600 MHz, CDCl₃) 8.59 (s, 1H, NH), 8.22-8.16 (m, 2H, 2×Ar—H), 7.66(d, 1H, J=7.5, Ar—H), 7.56 (t, 1H, J=8.0, H-11), 4.14 (t, 1H, J=8.6,H-5), 3.23 (dd, 1H, J=3.0 and 11.6, H-7), 3.05 (ddd, 1H, J=8.5, 11.1 and13.1, HH-6), 2.73 (ddd, 1H, J=3.6, 9.0 and 12.9, HH-6), 2.45 (ddd, 1H,J=6.7, 8.1 and 11.4, HH-19), 2.13 (ddd, 1H, J=6.8, 8.0 and 11.3, HH-19),1.30-1.24 (m, 2H, H₂-18), 1.21-1.08 (m, 6H, H₂-15, H₂-16 and H₂-17),0.81 (t, 3H, J=7.1, H₃-14); δ_(C) (150 MHz, CDCl₃) 177.94 (C═O), 176.84(C═O), 148.14 (C9), 138.76 (C13), 135.29 (Ar—H), 129.24 (Ar—H), 123.39(Ar—H), 123.16 (Ar—H), 56.71 (C4), 45.09 (C5), 43.69 (C7), 31.26 (CH₂),28.88 (CH₂), 28.84 (C19), 28.51 (C6), 26.33 (CH₂), 22.57 (CH₂), 14.06(C14); IR (oil, cm⁻¹) 3214 (w), 2928 (w) 1773 (m), 1709 (s); MS (CI+)m/z (relative intensity): 363 ([M+H], 10), 214 (15), 84 (100); ExactMass Calcd for [C₁₈H₂₂N₂O₄S]+H requires m/z 363.1379. Found 363.1394(CI+).

Reference Example 90: Preparation of (4RS, 5SR, 7RS)2-Aza-4-(N-Boc-L-Cys-OMe)-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

N-Boc-Cys(Mal)-OMe (50 mg, 0.15 mmol) was dissolved in acetonitrile (30mL) to provide a 0.005M solution. The resulting solution was degassedfor 30 minutes, styrene (136 μL, 1.2 mmol) added and irradiated in pyrexglassware for 5 minutes with stirring. Solvent was removed in vacuo andpurification by flash chromatography (gradient elution in petroleumether to 30% ethyl acetate in petroleum ether) afforded the desiredcompound as a thick colourless oil (16 mg, 0.037 mmol) in 24% yield as amixture of two major diastereomers (small signals suggest two otherdiastereomers, possibly regioisomers regarding the addition of thestyrene). Reanalysis of the crude suggests that the reaction wassuccessful in at least 80%. δ_(H) (600 MHz, CDCl₃) 8.08 (s, 2H, 2×H-2),7.40-7.31 (m, 10H, 10×Ar—H), 5.0 (d, 1H, J=8.2, H-15), 4.9 (d, 1H,J=7.5, H-15), 4.26-4.23 (m, 1H, H-16), 4.18-4.12 (m, 1H, H-16), 4.06 (t,2H, J=8.5, 2×H-5), 3.669 (s, 3H, H₃-18), 3.674 (s, 3H, H₃-18), 3.19(ddd, 1H, J=2.4 and 11.0, H-7), 3.11 (dd, 1H, J=3.2 and 11.0, H-7)3.04-2.93 (m, 3H, 2×HH-6 and HH-19), 2.91 (dd, 1H, J=6.6 and 12.8,HH-19), 2.64-2.60 (m, 2H, 2×HH-6), 2.51 (dd, 1H, J=4.6 and 12.8. HH-19),2.43 (dd, 1H, J=7.3 and 13.0, HH-19), 1.45 (s, 911, 3×H₃-12), 1.43 (s,9H, 3×H₃-12); δ_(C) (150 MHz, CDCl₃) 178.41 (C═O), 177.25 (C═O), 177.20(C═O), 171.40 (C═O), 171.10 (C═O), 170.98 (C═O), 155.28 (C═O), 155.18(C═O), 136.28 (C11), 136.25 (C1), 128.94 (2×Ar—H), 128.93 (2×Ar—H),128.49 (2×Ar—H), 128.46 (2×Ar—H), 128.38 (C8), 128.33 (C8), 80.44(2×C13), 56.71 (C4), 56.48 (C4), 53.03 (C16), 52.87 (C16), 52.78 (C18),52.75 (C18), 45.92 (C5), 45.82 (C5), 43.76 (C7), 43.61 (C7), 31.28 (C6),31.09 (C6), 28.38 (6×C12), 26.33 (C19), 26.21 (C19); IR (oil, cm⁻¹) 3215(w), 2971 (w) 1738 (s), 1715 (s); MS (CI+) m/z (relative intensity): 435([M+H], 10), 379 (30), 335 (100); Exact Mass Calcd for [C₂₁H₂₇N₂O₆S]+Hrequires m/z 435.1590. Found 435.1576 (CI+).

Reference Example 91: Preparation of1-(p-Methoxy)phenyl-4-Hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione,(4RS, 5RS, 7RS)2-Aza-4-Hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dioneand (4RS, 5SR, 7RS)2-Aza-4-Hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile(25 mL) to provide a 0.005M solution. The resulting solution wasdegassed for 30 minutes, 4-methoxy styrene (154 μL, 1.20 mmol) added andirradiated in pyrex glassware for 5 minutes with stirring. Solvent wasremoved in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded1-(p-methoxy)phenyl-4-Hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione asa colourless oil (10 mg, 0.037 mmol) in 25% yield and (4RS, 5RS, 7RS)2-aza-4-hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dione(major) and (4RS, 5SR, 7RS)2-aza-4-hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dione(minor) as a colourless oil (27 mg, 0.77 mmol) in 67% yield as a mixtureof diastereomers (10:1).

4-Hexylsulfanyl-1-(p-methoxy)phenyl-1,7-dihydro-2H-azepine-3,6-dione

δ_(H) (600 MHz, CDCl₃) 7.21 (d, 2H, J=8.5, 2×Ar—H), 7.18 (s, 1H, NH),6.84 (d, 2H, J=9.0, 2×Ar—H), 6.13 (s, 1H, H-5), 4.08 (t, 1H, J=7.9,H-1), 3.80 (s, 3H, H₃-18), 2.99 (dd, 1H, J=7.2 and 15.7. HH-7), 2.91(dd, 1H, J=8.7 and 15.7, HH-7), 2.35-2.26 (m, 2H, H₂-13), 1.51-1.43 (m,2H, H₂-12), 1.33-1.16 (m, 6H. H₂-9H₂-10 and Hz-11), 0.85 (t, 3H, J=7.0,H₃-8); δ_(C) (150 MHz, CDCl₃) 171.03 (C═O), 170.05 (C═O), 159.06 (C17),147.37 (C4), 132.92 (C14), 129.37 (C5), 128.79 (2×Ar—H), 114.18(2×Ar—H), 55.38 (C18), 46.61 (C1), 32.59 (C7), 31.45 (C13), 31.38 (CH₂),29.22 (CH₂), 28.64 (CH₂), 22.61 (CH₂), 14.14 (C8); IR (oil, cm⁻¹) 3275(w), 2927 (m) 1774 (w), 1717 (s); MS (CI+) m/z (relative intensity): 347([M+], 15), 237 (70), 230 (100), 202 (60); Exact Mass Calcd for[C₁₉H₂₅NO₃S]+ requires m/z 347.1550. Found 363.1553 (CI+).

(4RS, 5RS, 7RS)2-Aza-4-hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dione(in bold) and (4RS, 5SR, 7RS)2-Aza-4-hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (600 MHz, CDCl₃) 8.71 (s, 1H, NH), 8.45 (s, 0.1H, NH), 7.24 (d,2H, J=7.86 2×Ar—H), 7.15 (d, 0.2H, J=8.7, 2×Ar—H), 6.90 (d, 1H, J=8.6,2×Ar—H), 6.86 (d, 0.2H, J=8.6, 2×Ar—H), 3.99 (t, 1H, J=8.8, H-5), 3.94(dd, 0.1H, J=8.4 and 10.1, H-5), 3.81 (s, 3H, H₃-8), 3.77 (s, 0.3H,H₃-8), 3.20 (dd, 0.111, J=4.5 and 11.9, H-7), 3.13 (dd, 1H, J=3.4 and11.0, H-7), 3.09 (dt, 0.1H, J=10.6 and 13.2, HH-6), 2.98 (ddd, 1H,J=8.7, 11.2 and 12.9, HH-6), 2.67 (dt, 0.1H, J=7.3 and 11.5, HH-18),2.63-2.53 (m, 1.1H, HH-6 and HH-18), 2.54 (ddd, 1H, J=4.5, 8.4 and 11.9,HH-6), 2.43 (ddd, 1H, J=6.7, 8.2 and 11.3, HH-18), 2.15 (ddd, 1H, J=6.7,8.4 and 11.4, HH-18), 1.56-1.52 (m, 0.2H, H₂-17), 1.39-1.33 (m, 0.2H,CH₂), 1.31-1.09 (m, 8.4H, H₂-14, H₂-15, H₂-16, H₂-17 and 2×CH₂) 0.87 (t,0.3H, J=7.1, H₃-13), 0.83 (t, 3H, J=7.1, H₃-13); Only signals of majordiastereoisomer shown δ_(C) (150 MHz, CDCl₃) 177.81 (C═O), 175.48 (C═O),159.39 (C9), 129.94 (2×Ar—H), 128.56 (C12), 113.65 (2×Ar—H), 57.63 (C4),55.38 (C8), 45.25 (C5), 43.78 (C7), 31.33 (CH₂), 29.23 (CH₂), 29.01(CH₂), 28.62 (CH₂), 26.53 (C18), 22.52 (C6), 14.10 (C13); IR (oil, cm⁻¹)3216 (w), 2928 (m) 1771 (m), 1707 (s); MS (CI+) m/z (relativeintensity): 348 ([M+H], 20), 135 (20), 134 (100); Exact Mass Calcd for[C₁₉H₂₅NO₃S]+H requires m/z 348.1633. Found 363.1642 (CI+).

Reference Example 92: Preparation of (4RS, 5SR, 7RS)2-Aza-4-(N-Ac-L-Cys-Benzylamine)-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

N-Ac-Cys(Mal)-Benzylamine (29 mg, 0.084 mmol) was dissolved inacetonitrile (50 mL) to provide a 0.002M solution. The resultingsolution was degassed for 30 minutes, styrene (10 μL, 0.084 mmol) addedand irradiated in pyrex glassware for 5 minutes with stirring. Solventwas removed in vacuo and purification by flash chromatography (gradientelution in 30% ethyl acetate in petroleum ether to 10% methanol in ethylacetate) afforded the desired compound as a colourless oil (33 mg, 0.073mmol) in 87% as a mixture of diastereomers of the [2+2] reaction.Reanalysis of the crude suggests the reaction was successful in around70%.

N-Ac-Cys(Mal)-Benzylamine (58 mg, 0.17 mmol) was dissolved inacetonitrile (80 mL) to provide a 0.002M solution. The resultingsolution was degassed for 30 minutes, styrene (191 μL, 1.70 mmol) addedand irradiated in pyrex glassware for 5 minutes with stirring. Solventwas removed in vacuo and purification by flash chromatography (gradientelution in 30% ethyl acetate in petroleum ether to 10% methanol in ethylacetate) afforded the desired compound as a colourless oil (14 mg, 0.031mmol) in 19% yield. Reanalysis of the crude suggests the reaction wassuccessful in at least 75%.

δ_(H) (600 MHz, CDCl₃) 8.69 (s, 1H, H-2), 7.36-7.26 (m, 8H, 8×Ar—H),7.18 (d, 2H, J=7.0, 2×Ar—H), 6.65 (t, 1H, J=5.6, H-17), 6.51 (d, 1H,J=7.4, H-14), 4.33 (d, 2H, J=6.0, H₂-18), 4.30 (td, 1H, J=1.2 and 5.4,H-15), 4.05 (t, 1H, J=8.9, H-5), 3.16 (dd, 1H, J=3.1 and 11.1, H-7),3.04 (ddd, 1H, J=8.9, 11.1 and 12.8. HH-23), 2.94 (dd, 1H, J=6.6 and13.3. HH-6), 2.59 (ddd, 1H, J=3.4, 8.9 and 12.5, HH-23), 2.31 (dd, 1H,J=5.3 and 13.6, HH-6), 1.98 (s, 3H, H₃-12); δ_(C) (150 MHz, CDCl₃)179.07 (C═O), 176.97 (C═O), 171.10 (C═O), 169.67 (C═O), 137.67 (Ar),136.16 (Ar), 129.02 (2×Ar—H), 128.80 (2×Ar—H), 128.55 (2×Ar—H), 128.35(Ar—H), 128.78 (2×Ar—H), 127.66 (Ar—H), 57.44 (C4), 52.61 (C15), 46.09(C5), 43.68 (C7), 43.66 (C18), 30.58 (C6), 26.15 (C23), 23.17 (C12); IR(oil, cm⁻¹) 3437 (w), 1726 (s); MS (FAB+) m/z (relative intensity): 695([M+H], 10), 439 (10), 286 (100); Exact Mass Calcd for [C₃₂H₃₄N₆O₈S₂]+Hrequires m/z 695.1958. Found 695.1964 (FAB+).

Reference Example 93: Preparation of (4RS, 5RS, 7RS)2-Aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dioneand (4RS, 5SR, 7RS)2-Aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

N-Methyl hexylsulfanylmaleimide (27 mg, 0.119 mmol) was dissolved inacetonitrile (25 mL) to provide a 0.005M solution. The resultingsolution was degassed for 30 minutes, styrene (136 μL, 1.19 mmol) addedand irradiated in pyrex glassware for 5 minutes with stirring. Solventwas removed in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded (4RS, 5RS, 7RS)2-aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dioneas a colourless oil (5 mg, 0.015 mmol) in 13% and (4RS, 5SR, 7RS)2-aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dioneas a colourless oil (23 mg, 0.069 mmol) in 58% yield.

(4RS, 5RS, 7RS)2-Aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (600 MHz, CDCl₃) 7.32-7.30 (m, 3H, 3×Ar—H), 7.13 (d, 2H, J=7.9,2×H-10), 3.99 (dd, 1H, J=7.8 and 10.2, H-5), 3.20 (dd, 1H, J=4.8 and10.3, H-7), 3.13-3.07 (m, 1H, HH-6), 2.93 (s, 3H, H₃-18), 2.65 (td, 1H,J=7.5 and 11.4, HH-17), 2.58 (td, 1H, J=7.5 and 11.8. HH-17), 2.46 (ddd,1H, J=4.9, 7.7 and 12.0, HH-6), 1.55-1.25 (m, 8H, H₂-13, H₂-14, H₂-15and H₂-16), 0.87 (t, 3H, J=7.0, H₃-12); δ_(C) (150 MHz, CDCl₃) 177.78(C═O), 175.17 (C═O), 137.21 (C11), 128.71 (2×Ar—H), 127.93 (C8), 127.24(2×Ar—H), 48.23 (C5), 42.71 (C7), 31.42 (CH₂), 30.07 (C17), 29.33 (CH₂),28.69 (CH₂), 25.71 (C12), 25.25 (C18), 22.58 (CH₂), 14.12 (C12); IR(oil, cm⁻¹) 2927 (w) 1715 (s); MS (CI+) m/z (relative intensity): 332([M+H], 40), 228 (40), 86 (70), 84 (100); Exact Mass Calcd for[C₁₉H₂NO₂S]+H requires m/z 332.1684. Found 333.1697 (CI+).

(4RS, 5SR, 7RS)2-Aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (600 MHz, CDCl₃) 7.39-7.30 (m, 5H, 5×Ar—H), 3.90 (t, 1H, J=8.7,H-5) 3.13-3.11 (m, 4H, H₃-18 and H-7), 3.00 (ddd, 1H, J=8.5, 11.0 and12.8. HH-6), 2.53 (ddd, 1H, J=3.7, 9.1 and 12.8, HH-6), 2.40 (ddd, 1H,J=6.4, 8.1 and 11.3, HH-17), 2.06 (ddd, 1H, J=6.5, 8.3 and 11.3, HH-17),1.25-1.08 (m, 8H, H₂-13, H₂-14, H₂-15 and H₂-16), 0.82 (t, 3H, J=7.4,H₃-12); δ_(C) (150 MHz, CDCl₃) 178.73 (C═O), 177.70 (C═O), 136.77 (C11),128.89 (2×Ar—H), 128.27 (2×Ar—H), 127.99 (C8), 55.69 (C4), 45.62 (C5),42.61 (C7), 31.29 (CH₂), 28.94 (CH₂), 28.68 (CH₂), 28.55 (C17), 26.26(C6), 25.70 (C18), 22.58 (CH₂), 14.09 (C12); IR (oil, cm⁻¹) 2927 (w)1703 (s); MS (CI+) m/z (relative intensity): 332 ([M+H], 100); ExactMass Calcd for [C₁₉H₂₅NO₂S]+H requires m/z 332.1684. Found 332.1680(CI+).

Reference Example 94: Preparation of (4RS, 5RS, 7RS)2-Aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione,(4RS, 5SR, 7RS)2-Aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dioneand 1-Phenyl-3-phenyl-4-hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione

N-Phenyl hexylsulfanylmaleimide (34 mg, 0.12 mmol) was dissolved inacetonitrile (25 mL) to provide a 0.005M solution. The resultingsolution was degassed for 30 minutes, styrene (135 μL, 1.18 mmol) addedand irradiated in pyrex glassware for 5 minutes with stirring. Solventwas removed in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded (4RS, 5RS, 7RS)2-aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dioneand (4RS, 5SR, 7RS)2-aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dioneas a colourless oil (37 mg, 0.94 mmol) in 80% yield as a mixture ofdiastereoisomers (11:2) and1-phenyl-3-phenyl-4-hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione as acolourless oil (0.5 mg, 0.001 mmol) in 1% yield (4RS, 5RS, 7RS)2-aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dioneand (4RS, 5SR, 7RS)2-aza-2-phenyl-4-hexylsulfanyl-S-phenyl-bicyclo[3.2.0]heptan-1,3-dione

(4RS, 5RS, 7RS)2-Aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dionein Bold

δ_(H) (600 MHz, CDCl₃) 7.54-7.52 (m, 11H, 2×Ar—H), 7.46-7.42 (m, 4H,4×Ar—H), 7.41-7.37 (m, 46H, 8×Ar—H and 2×Ar—H), 7.27-7.26 (m, 2H,2×Ar—H), 7.06-7.05 (m, 2H, 2×Ar—H), 4.09 (t, 1H, J=8.5, H-5), 4.08 (t,5.5H, J=8.5, H-5), 3.35 (dd, 1H, J=4.7 and 10.5, H-7), 3.29 (dd, 5.5H,J=4.0 and 10.9, H-7), 3.20 (td, 1H, J=10.3 and 13.3, HH-6), 3.09 (ddd,5.5H, J=8.2, 11.0 and 13.0, HH-6), 2.78 (td, 1H, J=7.4 and 11.7, HH-17),2.72-2.68 (m, 6.5H, HH-6 and HH-6), 2.66 (dd, 1H, J=4.8 and 7.4, HH-17),2.52 (ddd, 5.5H, J=6.5, 8.2 and 11.4, HH-17), 2.16 (ddd, 5.5H, J=6.6,8.4 and 11.4, HH-17), 1.63-1.58 (m, 2H, H₂-16), 1.42-1.37 (m, 2H.H₂-15), 1.36-1.09 (m, 24H, 4×CH₂ and 2×CH₂), 0.88 (t, 3H, J=6.7, H₃-12),0.83 (t, 16.5H, J=7.3, H₃-12); δ_(C) (150 MHz, CDCl₃) 177.65 (C═O),176.82 (C═O), 176.67 (C═O), 174.08 (C═O), 136.96 (C11), 136.82 (C11),132.06 (C21), 131.86 (C21), 129.40 (2×Ar—H), 129.22 (Ar—H), 128.96(2×Ar—H), 128.84 (Ar—H), 128.79 (Ar—H), 128.34 (2×Ar—H), 128.08 (Ar—H),127.51 (Ar—H), 126.56 (2×Ar—H), 126.34 (Ar—H), 55.57 (C4), 55.47 (C4),48.78 (C5), 45.87 (C5), 44.88 (C7), 42.79 (C7), 31.45 (C17), 31.31(C17), 30.27 (CH₂), 29.46 (CH₂), 29.08 (CH₂), 28.75 (CH₂), 28.56 (CH₂),26.95 (CH₂), 22.60 (CH₂), 22.52 (CH₂), 14.14 (C12), 14.10 (C12) Severalcarbon signals are missing due to overlap of the diastereomers; IR (oil,cm⁻¹) 2926 (w) 1709 (s); MS (CI+) m/z (relative intensity): 394 ([M+H],70), 290 (100), 105 (100); Exact Mass Calcd for [C₂₄H₂₇NO₂S]+H requiresm/z 394.1841. Found 394.1834 (CI+).

1-Phenyl-3-phenyl-4-hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione

δ_(H) (600 MHz, CDCl₃) 7.54-7.27 (m, 10H, 10×Ar—H), 6.32 (s, 1H, H-6),4.19 (t, 1H, J=88.0, H-1), 3.14-3.03 (m, 2H, H₂-2), 2.39-2.29 (m, 2H,H₂-13), 1.61-1.10 (m, 8H, 4×CH₂), 0.89-0.81 (m, 3H, H₃-8); δ_(C) (150MHz, CDCl₃) 146.31 (C═O), 141.19 (C═O), 130.19 (Ar), 129.41 (Ar), 129.20(Ar—H), 128.93 (2×Ar—H), 128.49 (2×Ar—H), 127.90 (Ar—H), 127.76(2×Ar—H), 126.03 (2×Ar—H), 47.19 (C1), 32.70 (CH₂), 31.43 (CH₂), 29.83(CH₂), 29.20 (CH₂), 28.63 (CH₂), 22.61 (CH₂), 14.13 (C8); IR (oil, cm⁻¹)2926 (m) 1715 (s); MS (CI+) m/z (relative intensity): 394 ([M+H], 40),278 (100); Exact Mass Calcd for [C₂₄H₂₇NO₂S]+H requires m/z 394.1841.Found 394.1829 (CI+).

Reference Example 95: Preparation of (4RS, 5SR, 7RS)2-Aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione and (4RS,5RS, 7RS) 2-Aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

Phenylthiomaleimide (17 mg, 0.082 mmol) was dissolved in acetonitrile(25 mL) to provide a 0.003M solution. The resulting solution wasdegassed for 30 minutes, styrene (111 μL, 0.82 mmol) added andirradiated in pyrex glassware for 5 minutes with stirring. Solvent wasremoved in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded (4RS, 5SR, 7RS)2-aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione as acolourless oil (1.5 mg, 0.005 mmol) in 6% yield and (4RS, 5RS, 7RS)2-aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione as acolourless oil (17.5 mg, 0.056 mmol) in 69% yield.

(4RS, 5SR, 7RS)2-Aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (600 MHz, CDCl₃) 8.05 (s, 1H, NH), 7.42-7.41 (m, 2H, 2×Ar—H),7.35-7.17 (m, 8H, 8×Ar—H), 4.05 (t, 1H, J=10.1, H-5), 3.29 (dd, 1H,J=5.5 and 13.0, H-7), 3.01 (dt, 1H, J=10.3 and 13.0, HH-6), 2.56 (ddd,1H, J=5.6, 10.1 and 13.4, HH-6); δ_(C) (150 MHz, CDCl₃) 176.69 (C═O),174.10 (C═O), 136.56 (C1), 136.01 (2×Ar—H), 130.13 (Ar—H), 129.66(2×Ar—H), 129.28 (C15), 128.72 (2×Ar—H), 128.01 (Ar—H), 127.32 (2×Ar—H),60.59 (C4), 46.32 (C5), 43.70 (C7), 25.51 (C6); IR (oil, cm⁻¹) 3226 (w),2925 (w) 1715 (s); MS (CI+) m/z (relative intensity): 310 ([M+H], 10),206 (30), 111 (100); Exact Mass Calcd for [C₁₈H₁₅NO₂S]+H requires m/z310.0902. Found 310.0901 (CI+).

(4RS, 5RS, 7RS)2-Aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (600 MHz, CDCl₃) 8.24 (s, 1H, NH), 7.43-7.30 (m, 6H, 8×Ar—H),7.27-7.24 (m, 2H, 2×Ar—H), 4.13 (t, 1H, J=9.0, H-5), 3.20-3.13 (m, 2H,HH-6 and H-7), 2.55 (ddd, 1H, J=5.6, 8.3 and 13.4, HH-6); δ_(C) (150MHz, CDCl₃) 177.99 (C═O), 177.17 (C═O), 135.80 (C11), 135.80 (2×Ar—H),129.64 (Ar—H), 129.54 (2×Ar—H), 128.95 (C15), 128.51 (2×Ar—H), 128.45(2×Ar—H), 128.22 (Ar—H), 60.72 (C4), 45.80 (C5), 43.39 (C7), 25.20 (C6);IR (oil, cm⁻¹) 3211 (w), 1772 (w) 1707 (s); MS (CI+) m/z (relativeintensity): 310 ([M+H], 50), 206 (100), 104 (40); Exact Mass Calcd for[C₁₈H₁₅NO₂S]+H requires m/z 310.0902. Found 310.0905 (CI+).

Reference Example 96: Preparation of (4RS, 7RS)2-Aza-4-hexylsulfanyl-bicyclo[3.2.0]hept-5-ene-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile(25 mL) to provide a 0.005M solution. The resulting solution wasdegassed for 30 minutes, phenyl acetylene (128 μL, 1.16 mmol) added andirradiated in pyrex glassware for 30 minutes with stirring. Solvent wasremoved in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded the desired compound as a colourless oil (4.5 mg, 0.014 mmol)in 18% yield (based on recovered SM) alongside SM (1.5 mg, 0.007 mmol)in 6% yield. δ_(H) (600 MHz, CDCl₃) 7.86 (s, 1H, NH), 7.68 (d, 2H,J=7.86, 2×Ar—H), 7.40-7.34 (m, 3H, 3×Ar—H), 6.58 (s, 1H, H-5), 3.73 (s,1H, H-7), 2.57 (dt, 1H, J=2.3 and 7.4, H₂-13), 1.77-1.19 (m, 8H, 4×CH₂),0.85 (t, 3H, J=7.3, H₃-8); δ_(C) (150 MHz, CDCl₃) 173.46 (C═O), 173.28(C═O), 149.72 (C17), 130.25 (C6), 130.02 (C14), 128.85 (2×Ar—H), 126.32(2×Ar—H), 125.76 (C5), 53.78 (C7), 31.33 (CH₂), 29.70 (CH₂), 29.32(CH₂), 28.61 (CH₂), 22.55 (CH₂), 14.11 (C8); IR (oil, cm⁻¹) 3228 (w),2925 (m), 1770 (w) 1709 (s); MS (CI+) m/z (relative intensity): 316([M+H], 100), 214 (30); Exact Mass Calcd for [C₁₈H₂₂NO₂S]+H requires m/z316.0371. Found 316.1365 (CI+).

Reference Example 97: Preparation of1-Butyl-4-hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione and (4RS, 5SR,7RS) 2-Aza-4-hexylsulfanyl-5-butyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.116 mmol) was dissolved in acetonitrile(20.7 mL) and hex-1-ene (4.3 mL, 11.6 mmol) to provide a 0.005Msolution. The resulting solution was degassed for 30 minutes andirradiated in pyrex glassware for 5 minutes with stirring. Solvent wasremoved in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded 1-butyl-4-hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione as acolourless oil (4 mg, 0.013 mmol) in 12% yield (alongside (4RS, 5SR,7RS) 2-aza-4-hexylsulfanyl-5-butyl-bicyclo[3.2.0]heptan-1,3-dione) and(4RS, 5SR, 7RS)2-aza-4-hexylsulfanyl-5-butyl-bicyclo[3.2.0]heptan-1,3-dione as acolourless oil (16 mg, 0.054 mmol) in 47% yield.

1-Butyl-4-hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione

δ_(H) (600 MHz, CDCl₃) 7.29 (s, 1H, NH), 6.43 (s, 1H, H-5), 2.91-2.76(m, 3H, H-1 and H₂-17), 2.70 (dd, 1H, J=1.4 and 5.8, HH-7), 2.64 (dd,1H, J=1.4 and 7.9, HH-7), 2.49 (t, 1H, J=7.4, H₂-13), 1.81-1.25 (m, 12H,6×CH₂), 0.93-0.85 (m, 6H, H₃-8 and H₃-14); δ_(C) (150 MHz, CDCl₃) 174.39(C═O), 171.40 (C═O), 148.19 (C4), 129.28 (C5), 43.90 (C1), 34.97 (CH₂),31.53 (CH₂), 30.60 (CH₂), 29.70 (CH₂), 29.07 (CH₂), 28.76 (CH₂), 28.51(CH₂), 22.66 (CH₂), 22.65 (CH₂), 14.16 (CH₃), 14.13 (CH₃); IR (oil,cm⁻¹) 3226 (w), 2927 (m) 1715 (s); MS (CI+) m/z (relative intensity):298 ([M+H], 80), 187 (100); Exact Mass Calcd for [C₁₆H₂₇NO₂S]+H requiresm/z 298.1841. Found 298.1841 (CI+).

(4RS, 5SR, 7RS)2-Aza-4-hexylsulfanyl-5-butyl-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (600 MHz, CDCl₃) 8.19 (s, 1H, NH), 3.09 (dd, 1H, J=4.5 and 10.2,H-7), 2.70-2.64 (m, 1H, H-5), 2.53-2.45 (m, 2H, H₂-17), 2.37-2.28 (m,2H, H₂-6), 1.80-1.73 (m, 1H, HH-11), 1.59-1.50 (m, 2H. HH-11 and HH-10),1.38-1.20 (m, 11H, HH-10 and 5×CH₂), 0.90 (t, 3H, J=7.1, CH₃), 0.87 (t,3H, J=7.3, CH₃); δ_(C) (150 MHz, CDCl₃) 178.73 (C═O), 177.81 (C═O),56.10 (C4), 44.29 (C7), 40.99 (C5), 31.42 (C17), 29.24 (CH₂), 28.99(CH₂), 28.90 (CH₂), 28.74 (CH₂), 28.73 (CH₂), 27.92 (CH₂), 22.59 (CH₂),22.58 (CH₂), 14.12 (CH₃), 14.09 (CH₃); IR (oil, cm⁻¹) 3209 (w), 2927 (m)1774 (w), 1711 (s); MS (CI+) m/z (relative intensity): 298 ([M+H], 100);Exact Mass Calcd for [C₁₆H₂₇NO₂S]+H requires m/z 298.1841. Found298.1845 (CI+).

Reference Example 98: Preparation of (4RS, 7RS)2-Aza-4-hexylsulfanyl-5-ethyl-6-ethyl-bicyclo[3.2.0]hept-5-ene-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.116 mmol) was dissolved in acetonitrile(21.1 mL) and hex-3-yne (3.9 mL, 11.6 mmol) to provide a 0.005Msolution. The resulting solution was degassed for 30 minutes andirradiated in pyrex glassware for 5 minutes with stirring. Solvent wasremoved in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded the desired compound as a colourless oil (17 mg, 0.057 mmol) in49% yield. δ_(H) (600 MHz, CDCl₃) 8.26 (s, 1H, NH), 3.88 (s, 1H, H-7),2.99 (ddd, 1H, J=7.5, 9.1 and 12.8, HH-18), 2.87 (ddd, 1H, J=5.1, 8.7and 12.9, HH-18), 2.39-2.20 (m, 2H, HH-9 and HH-11), 1.95-1.75 (m, 2H,HH-9 and HH-11), 1.56-1.10 (m, 8H, 4×CH₂), 0.90-0.86 (m, 9H, H₃-8, H₃-10and H₃-12); δ_(C) (150 MHz, CDCl₃) 172.66 (C═O), 171.34 (C═O), 148.84(C═C), 142.53 (C═C), 70.45 (C4), 48.86 (C18), 48.09 (C7), 31.45 (CH₂),28.51 (CH₂), 23.53 (CH₂), 22.50 (CH₂), 21.89 (CH₂), 21.62 (CH₂) 14.09(C12), 12.03 (CH₃), 11.84 (CH₃); IR (oil, cm⁻¹) 2931 (m) 1717 (s); MS(CI+) m/z (relative intensity): 312 ([M+OH], 100), 178 (100); Exact MassCalcd for [C₁₆H₂₅NO₂S]+OH requires m/z 312.1633. Found 312.1648 (CI+).

Reference Example 99: Preparation of1-Ethyl-2-ethyl-6-hexylsulfanyl-1,2-dihydro-3H-azepine-4,7-dione

Hexylsulfanylmaleimide (25 mg, 0.116 mmol) was dissolved in acetonitrile(21.1 mL) and trans-hex-3-ene (3.9 mL, 11.6 mmol) to provide a 0.005Msolution. The resulting solution was degassed for 30 minutes andirradiated in pyrex glassware for 5 minutes with stirring. Solvent wasremoved in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded the desired compound as a colourless oil (2 mg, 0.007 mmol) in6% yield. δ_(H) (600 MHz, CDCl₃) 7.32 (s, 1H, NH), 6.49 (s, 1H, H-6),2.92 (ddd, 1H, J=4.6, 6.3 and 10.8, H-1), 2.83 (ddd, 1H, J=5.2, 9.4 and12.8, HH-13), 2.75 (dd, 1H, J=5.9 and 10.9, H-2), 2.62 (ddd, 1H, J=6.7,9.6 and 12.7 HH-13), 2.07-2.00 (m, 1H, HH-17), 1.89-1.83 (m, 1H, HH-15),1.79-1.40 (m, 6H, HH-15, HH-17 and 2×CH₂), 1.33-1.30 (m, 4H, 2×CH₂),1.10 (t, 3H, J=7.5. H₃-16), 0.92 (t, 3H, J=7.4, CH₃), 0.89 (t, 3H,J=7.0, CH₃); δ_(C) (150 MHz, CDCl₃) 171.00 (C═O), 169.53 (C═O), 149.76(C5), 129.27 (C6), 62.54 (C2), 51.16 (C13), 40.42 (C1), 31.48 (CH₂),28.65 (CH₂), 23.65 (CH₂), 23.32 (CH₂), 22.52 (CH₂), 17.23 (C17) 14.11(CH₃), 13.96 (CH₃), 12.33 (CH₃); IR (oil, cm⁻¹) 2962 (m) 1717 (s); MS(CI+) m/z (relative intensity): 314 ([M+OH], 75), 180 (100); Exact MassCalcd for [C₁₆H₂₆NO₂S]+OH requires m/z 314.1790. Found 314.1799 (CI+).

Reference Example 100: Preparation of (4RS, 7RS)2-Aza-4-hexylsulfanyl-5,5-diphenyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.116 mmol) was dissolved in acetonitrile(21 mL) and 1,1-diphenylethyene (203 μL, 1.16 mmol) to provide a 0.005Msolution. The resulting solution was degassed for 30 minutes andirradiated in pyrex glassware for 5 minutes with stirring. Solvent wasremoved in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded the desired compound as a colourless oil (30 mg, 0.075 mmol) in64% yield. δ_(H) (600 MHz, CDCl₃) 8.15 (s, 1H, NH), 7.42 (m, 2H, 2×H-8),7.36-7.21 (m, 8H, 8×Ar—H), 3.54 (dd, 1H, J=10.3 and 12.9, HH-6), 3.34(dd, 1H, J=5.7 and 10.3, H-7), 3.18 (dd, 1H, J=5.8 and 12.9, HH-6), 2.43(dt, 1H, J=7.3 and 11.0, HH-17), 2.34 (dt, 1H, J=7.4 and 11.0, HH-17),1.40-1.34 (m, 2H, H₂-16), 1.26-1.20 (m, 4H, H₂-14 and H₂-15), 1.18-1.13(m, 2H, H₂-13), 0.84 (t, 3H, J=7.5, H₃-12); δ_(C) (150 MHz, CDCl₃)177.16 (C═O), 175.87 (C═O), 142.09 (2×Ar), 141.80 (2×Ar), 128.17(2×Ar—H), 128.13 (2×Ar—H), 128.10 (2×Ar—H), 128.06 (2×Ar—H), 127.44(Ar—H), 127.32 (Ar—H), 63.03 (C5), 57.20 (C4), 44.38 (C7), 35.17 (C6),31.35 (CH₂), 30.07 (C17), 28.77 (CH₂), 28.71 (CH₂) 22.53 (CH₂), 14.11(C12); IR (oil, cm⁻¹) 2927 (m) 1772 (w), 1709 (s); MS (ES−) m/z(relative intensity): 392 ([M], 10), 212 (100); Exact Mass Calcd for[C₂₄H₂₆NO₂S] requires m/z 392.1684. Found 392.1674 (ES−).

Reference Example 101: Preparation of (4RS, 5RS, 7RS)2-Aza-2-methylenecyclohexane-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dioneand (4RS, 5SR, 7RS)2-Aza-2-methylenecyclohexane-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

N-Methylene hexylsulfanylmaleimide (25 mg, 0.116 mmol) was dissolved inacetonitrile (21 mL) and 1,1-diphenylethyene (203 μL, 1.16 mmol) toprovide a 0.005M solution. The resulting solution was degassed for 30minutes and irradiated in pyrex glassware for 5 minutes with stirring.Solvent was removed in vacuo and purification by flash chromatography(gradient elution in petroleum ether to 30% ethyl acetate in petroleumether) afforded (4RS, 5RS, 7RS)2-aza-2-methylenecydohexane-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dioneand (4RS, 5SR, 7RS)2-aza-2-methylenecyclohexane-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dioneas a colourless oil (30 mg, 0.075 mmol) as a mix of diastereoisomers(10:1) in 64% yield.

(4RS, 5RS, 7RS)2-aza-2-methylenecyclohexane-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dionein bold

δ_(H) (600 MHz, CDCl₃) 7.39-7.29 (m, 5.2H, 5×Ar—H and 0.2×Ar—H), 7.24(d, 0.1H, J=7.4, H-8), 7.16 (d, 2H, J=7.4, 0.2×H-10), 4.00 (dd, 0.1H,J=8.2 and 9.9, H-5), 3.89 (t, 1H, J=8.7, H-5), 3.46 (d, 2.2H, J=7.5,H₂-22 and H₂-22), 3.19 (dd, 0.1H, J=5.1 and 10.5, H-7), 3.12 (dd, 1H,J=3.5 and 10.9, H-7), 3.07 (td, 0.1H, J=10.5 and 13.1. HH-6), 3.00 (ddd,1H, J=8.5, 11.5 and 12.6, HH-6), 2.64 (ddd, 0.1, J=6.9, 11.4 and 14.8,HH-17), 2.56 (td, 0.1H, J=6.9 and 11.6. HH-6), 2.53 (ddd, 1H, J=3.3, 9.1and 12.9, HH-6), 2.48 (ddd, 0, 1H, J=5.4, 8.0 and 13.2, HH-17), 2.39(td, 1H, J=7.4 and 11.4, HH-17), 2.08 (td, 1H, J=7.7 and 11.4, HH-17),1.83-0.99 (m, 2H, 20.9H), 0.87 (t, 3H, J=7.0, H₃-12), 0.82 (t, 3H,J=7.5, H₃-12); δ_(C) (150 MHz, CDCl₃) 179.12 (C═O), 178.35 (C═O), 178.15(C═O), 175.41 (C═O), 137.23 (C11), 137.12 (C11), 129.08 (2×Ar—H), 128.76(2×Ar—H), 128.47 (2×Ar—H), 128.16 (C8), 127.99 (C8), 127.51 (2×Ar—H),55.84 (C4), 55.69 (C4), 48.06 (C5), 45.97 (C5), 45.63 (C22), 45.48(C22), 42.94 (C7), 42.83 (C7), 36.67 (C21), 36.58 (C21), 31.64 (CH₂),31.49 (CH₂), 31.08 (CH₂), 31.01 (CH₂), 30.96 (CH₂), 30.34 (CH₂), 30.04(CH₂), 29.67 (CH₂), 29.28 (CH₂), 28.98 (CH₂), 28.82 (CH₂), 26.76 (CH₂),26.63 (CH₂), 26.52 (CH₂), 26.45 (CH₂), 25.96 (CH₂), 25.94 (CH₂), 25.82(CH₂), 14.34 (C12), 14.31 (C12) Several carbon signals are missing dueto overlap of the diastereomers; IR (oil, cm⁻¹) 2925 (m) 1703 (s); MS(CI+) m/z (relative intensity): 414 ([M+H], 100), 309 (20); Exact MassCalcd for [C₂₅H₃₅NO₂S]+H requires m/z 414.2461. Found 414.2452 (CI+).

Reference Example 102: Preparation of (4RS, 5SR, 7SR)2-Aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dioneand (4RS, 5RS, 7SR)2-Aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dione

2,3 Dihexylsulfanylmaleimide (38 mg, 0.115 mmol) was dissolved inacetonitrile (25 mL) to provide a 0.005M solution. The resultingsolution was degassed for 30 minutes, styrene (133 μL, 1.2 mmol) addedand irradiated in pyrex glassware for 20 minutes with stirring. Solventwas removed in vacuo and purification by flash chromatography (gradientelution in petroleum ether to 30% ethyl acetate in petroleum ether)afforded (4RS, 5SR, 7SR)2-aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dioneas a colourless oil (3 mg, 0.007 mmol) in 6% yield and (4RS, 5RS, 7SR)2-aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dioneas a colourless oil (3 mg, 0.007 mmol) in 6% yield.

(4RS, 5SR, 7SR)2-Aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (600 MHz, CDCl₃) 7.96 (s, 1H, NH), 7.33 (d, 2H, J=7.0, 2×Ar—H),7.28 (t, 1H, J=7.0. H-8), 7.21 (d, 2H, J=7.5, 2×Ar-H), 4.02 (t, 1H,J=10.0, H-5), 2.98-2.92 (m, 2H, HH-6 and —S—CHH—), 2.87 (dd, 1H, J=10.8and 13.6, HH-6), 2.83-2.72 (m, 2H, —S—CHH— and —S—CHH—), 2.69 (dt, 1H,J=7.4 and 10.8, —S—CHH—) 1.68-1.57 (m, 4H, H₂-16 and H₂-22), 1.45-1.31(m, 4H, H₂-15 and H₂-21), 1.31-1.25 (m, 8H, HZ-13, HZ-14, H₂-19 andH₂-20), 0.86 (t, 6H, J=7.0, H₃-12 and H₃-18); δ_(C) (150 MHz, CDCl₃)176.91 (C═O), 172.96 (C═O), 136.09 (C1), 128.83 (2×Ar—H), 128.13 (C8),127.41 (2×Ar—H), 62.86 (C4), 54.36 (C7), 46.49 (C5), 33.28 (C6), 31.51(CH₂), 31.47 (CH₂), 30.65 (SCH₂), 30.09 (SCH₂), 29.22 (CH₂), 28.96(CH₂), 28.80 (CH₂), 28.74 (CH₂), 22.63 (CH₂), 22.60 (CH₂), 14.17 (CH₃),14.15 (CH₃); IR (oil, cm⁻¹) 3194 (w), 2928 (m) 1774 (w), 1722 (s); MS(CI+) m/z (relative intensity): 432 ([M−H], 5), 332 (50), 316 (95), 207(100); Exact Mass Calcd for [C₂₄H₃₅NO₂S₂]−H requires m/z 432.2026. Found432.2029 (CI+).

(4RS, 5RS, 7SR)2-Aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dione

δ_(H) (600 MHz, CDCl₃) 8.10 (s, 1H, NH), 7.41 (d, 2H, J=6.9, 2×Ar—H),7.37 (t, 1H, J=6.9, H-8), 7.33 (d, 2H, J=6.9, 2×Ar—H), 3.92 (t, 1H,J=8.9, H-5), 2.95 (dd, 1H, J=8.9 and 12.9, HH-6), 2.86 (dt, 1H, J=6.9and 14.2, —S—CHH—), 2.78-2.66 (m, 2H, HH-6 and —S—CHH—), 2.60 (ddd, 1H,J=6.3, 8.3 and 10.9, —S—CHH—) 2.00 (ddd, 1H, J=5.6, 8.6 and 10.7,—S—CHH—), 1.65-1.60 (m, 2H, HH-16 and HH-22), 1.43-1.06 (m, 14H, HH-16,HH-22, H₂-13, H₂-14, H₂-15, H₂-19, H₂-20 and H₂-21), 0.88 (t, 3H, J=6.7,CH₃), 0.82 (t, 3H, J=7.1, CH₃); δ_(C) (150 MHz, CDCl₃) 176.59 (C═O),176.44 (C═O), 136.03 (C11), 129.50 (2×Ar—H), 128.83 (C8), 128.29(2×Ar—H), 62.32 (C4), 54.58 (C7), 45.33 (C5), 34.85 (C6), 31.48 (CH₂),31.33 (CH₂), 30.51 (CH₂), 29.21 (CH₂), 29.06 (CH₂), 28.90 (CH₂), 28.76(CH₂), 28.53 (CH₂), 22.62 (CH₂), 22.50 (CH₂), 14.16 (CH₃), 14.11 (CH₃);IR (oil, cm⁻¹) 3215 (w), 2926 (m) 1774 (w), 1715 (s); MS (CI+) m/z(relative intensity): 432 ([M−H], 5), 329 (60), 207 (100), 161 (60);Exact Mass Calcd for [C₂₄H₃₅NO₂S₂]−H requires m/z 432.2026. Found432.2034 (CI+).

Example 40: Preparation of (4RS, 5RS, 7SR)2-Aza-4-(N-Boc-Cys-OMe)-5-phenyl-7-(N-Boc-Cys-OMe)-bicyclo[3.2.0]heptan-1,3-dione

2,3-Di-(N-Boc-Cys-OMe)-maleimide (76 mg, 0.135 mmol) was dissolved inacetonitrile (29 mL) to provide a 0.005M solution. The resultingsolution was degassed for 30 minutes, styrene (148 μL, 1.35 mmol) addedand irradiated in pyrex glassware for 30 minutes with stirring. Solventwas removed in vacuo and purification by flash chromatography (gradientelution in 10% ethyl acetate in petroleum ether to 30% ethyl acetate inpetroleum ether) afforded a mixture of (4RS, 5RS, 7SR)2-aza-4-(N-Boc-Cys-OMe)-5-phenyl-7-(N-Boc-Cys-OMe)-bicyclo[3.2.0]heptan-1,3-dionesand (4RS, 5SR, 7SR)2-aza-4-(N-Boc-Cys-OMe)-5-phenyl-7-(N-Boc-Cys-OMe)-bicyclo[3.2.0]heptan-1,3-dionesas a colourless oil (28 mg, 0.042 mmol) in 36% yield. The spectra fromthis mixture was very complex but MS confirmed the identity of thecompounds as all having the same mass, 4RS, 5RS, 7SR)2-aza-4-(N-Boc-Cys-OMe)-5-phenyl-7-(N-Boc-Cys-OMe)-bicyclo[3.2.0]heptan-1,3-dionesand (4RS, 5SR, 7SR)2-aza-4-(N-Boc-Cys-OMe)-5-phenyl-7-(N-Boc-Cys-OMe)-bicyclo[3.2.0]heptan-1,3-dioneswas also isolated alongside a [5+2] product as a colourless oil (46 mg)¹H NMR and MS data suggest 40% of this (by mass) is the desiredconjugation products (18 mg, 0.028 mmol) in 20% yield. δ_(H) (600 MHz,CDCl₃) 8.32 (d, 4.7H, J=8.7, N—H), 8.05 (s, 1.2H, N—H), 7.79 (s, 1H,N—H), 7.40-7.20 (m, 60H, Ar—H), 5.65 (d, 1H, J=7.2, H—N), 5.57 (d, 5.2H,J=8.2, H—N), 5.45 (d, 4.9H, J=7.3, H—N), 5.40 (d, 1.9H, J=7.8, H—N),4.92 (d, 2.5H, J=7.3, H-14), 4.74 (d, 3.7H, J=7.8, H-14), 4.62-4.54 (m,8H, H-14), 4.16-4.11 (m, 2.7H), 4.09-4.06 (m, 3.9H), 3.97-3.9 (m, 7.8H),3.80-3.75 (m, 50.5H, H₃-12), 3.66 (s, 22.6H, H₃-12) 3.47-3.04 (m, 41.4,H₂-18), 2.99-2.94 (m, 9.5H, H₂-18), 2.82-2.73 (m, 9.5H, H₂-18),1.46-1.42 (m, 240H, H₃-17); δ_(C) (150 MHz, CDCl₃) 175.94 (C═O), 175.84(C═O), 175.82 (C═O), 171.10 (C═O), 171.04 (C═O), 170.95 (C═O), 155.28(Ar), 129.63 (Ar—H), 129.50 (Ar—H), 128.96 (Ar—H), 128.88 (Ar—H), 128.80(Ar—H), 128.75 (Ar—H), 128.59 (Ar—H), 128.53 (Ar—H), 128.41 (Ar—H),80.51 (C16), 80.22 (C16), 52.97 (C12), 52.93 (C12), 52.71 (C14), 52.61(C14), 45.25 (C5), 32.92 (C18), 32.86 (C18), 31.19 (C18), 31.08 (C18),29.83 (C6), 28.42 (C17) Several carbon signals are missing due tooverlap of the diastereomers; IR (oil, cm⁻¹) 2924 (m), 1712 (s); MS(CI+) m/z (relative intensity): 666 ([M−H], 100); Exact Mass Calcd for[C₃H₄₁N₃O₁₀S₂]−H requires m/z 666.2155. Found 666.2188 (CI+).

The invention claimed is:
 1. A compound of formula (IIIa)

wherein: R₁ is an antibody or antibody fragment that is capable ofbinding to a specific antigen via an epitope on the antigen, saidantibody or antibody fragment containing a first cysteine residue and asecond cysteine residue; R₁ is attached at the 2-position in thecompound of formula (IIIa) by a thiol bond —S— which corresponds to thethiol group in said first cysteine residue; R₁ is attached at the3-position in the compound of formula (IIIa) by a thiol bond —S— whichcorresponds to the thiol group in said second cysteine residue; F₂ is adrug; and L represents a linker group.
 2. A compound according to claim1, wherein the first cysteine residue of R₁ and the second cysteineresidue of R₁ in the compound of formula (IIIa) are derived from aninternal disulfide bridge formed in the antibody or antibody fragment.3. A compound according to claim 1, wherein R₁ is an antibody.
 4. Acompound according to claim 1, wherein the drug is a cytotoxic agent. 5.A compound according to claim 3, wherein F₂ is a cytotoxic agent.